M.A. was supported by a Schr&#246;dinger fellowship of the Austrian Science Fund (FWF, J3086-B11) and thanks the Max-Planck-Society for financial and administrative support. G.S. and J.H. were supported by the Vienna Science and Technology Fund (WWTF, LS13-030).

<ol>
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D., Varma, R., Dustin, M. L., Vale, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+for+segregating+T+cell+receptor+and+adhesion+molecules+during+immunological+synapse+formation+in+Jurkat+T+cells.">Mechanisms for segregating T cell receptor and adhesion molecules during immunological synapse formation in Jurkat T cells.</a> Proc Natl Acad Sci USA. 104, 20296-20301 (2007).</li><li>Varma, R., Campi, G., Yokosuka, T., Saito, T., Dustin, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=T+cell+receptor-proximal+signals+are+sustained+in+peripheral+microclusters+and+terminated+in+the+central+supramolecular+activation+cluster.">T cell receptor-proximal signals are sustained in peripheral microclusters and terminated in the central supramolecular activation cluster.</a> Immunity. 25, 117-127 (2006).</li><li>Purbhoo, M. A., Irvine, D. J., Huppa, J. B., Davis, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=T+cell+killing+does+not+require+the+formation+of+a+stable+mature+immunological+synapse.">T cell killing does not require the formation of a stable mature immunological synapse.</a> Nat Immunol. 5, 524-530 (2004).</li><li>Binz, H. K., Stumpp, M. T., Forrer, P., Amstutz, P., Pluckthun, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Designing+repeat+proteins:+well-expressed,+soluble+and+stable+proteins+from+combinatorial+libraries+of+consensus+ankyrin+repeat+proteins.">Designing repeat proteins: well-expressed, soluble and stable proteins from combinatorial libraries of consensus ankyrin repeat proteins.</a> J Mol Biol. 332, 489-503 (2003).</li></ol>

The authors declare that they have no competing financial interest.

Measuring protein-protein interactions in situ is highly desirable especially when dealing with low affinity interactions such as TCR-pMHC binding 11. This is because the on-rate as well as the stability of such interactions are significantly influenced by the particular circumstances under which binding takes place. Minimally-invasive FRET-based imaging approaches are thus in principle perfectly suited for such tasks, yet involve a number of hurdles that must first be overcome. Noise generated by cellular autofluorescence limits the sensitivity of the measurements and should therefore be kept at a minimum. TIRF microscopy serves this need very well 12 but requires the functionalization of glass-slides, ideally in the form of a planar glass-supported lipid bilayer decorated with proteins of choice 13-15. Another advantage of a partly reconstitutive approach is that recombinant bilayer-resident FRET partners can be much more easily labeled in a quantitative, site-specific and rational manner with smaller and brighter fluorophores than would be possible with cell surface-expressed proteins. TCRs are tagged with recombinant scFVs, which do not influence T-cell recognition, as was tested previously 2. Moreover, the protein composition of the SLB, for example the density of pMHCs and the choice of accessory factors can be adjusted to one&#8217;s specific needs. We have previously performed experiments with varying densities of stimulatory pMHCs, but have not detected significant differences in 2D-koff and 2D-KD2.
So far here the recognition of MHC class II molecules has been dealt with only, mainly because of the nature of their peptide binding cleft, which is open at both ends and thus accommodates larger peptides including a linker for fluorophore attachment. In some cases such approach might also work for labeling MHC class I molecules 16 but great caution should be taken to verify their use in experiments. The sensitivity of T-cells towards antigens, which can be measured via T-cell proliferation assays, as well as the pMHC-TCR binding kinetics as measured in vitro by surface plasmon resonance should not be affected by the addition of the linker and fluorophore to the peptide. Alternatively, MHC class I molecules themselves can be labeled in a site-specific manner with the introduction of an unpaired cysteine within the sequence of the heavy chain (unpublished observations).
With the use of appropriate molecular probes any synaptic protein-protein interaction can in principle be studied in a fashion described herein. Such probes, e.g., scFVs or Designed Ankyrin Repeat Proteins (DARPins)17, should be monovalent and should bind their target stably without affecting the interaction of interest. Of course, structural information is highly desirable for rational probe design but not absolutely required. When establishing a new pair of FRET partners, it is recommended to record and analyze FRET in bulk first. Sites of label attachment may be varied considerably to maximize the FRET signal and also to verify that measured FRET yields differ based on the inter-dye distance. Once the system is optimized, single molecule FRET signals may be recorded by limiting the labeling of the high abundance FRET partner to 10-30% and bleaching the low abundance FRET partner until single molecules are resolvable in the field of illumination.
Last but not least it should be noted that SLBs approximate some but not all aspects of a physiological plasma membrane. Qualities such as membrane curvature and flexibility, domain compartmentalization, cytoskeletal rearrangements and cell motility as well as a high variety of surface-expressed membrane proteins are not represented by SLBs but might influence the process under investigation. Much effort will need to be invested to establish imaging modalities that allow monitoring protein-protein interactions with single molecule resolution in physiological synapses, which are inaccessible to TIRF imaging.

A more fundamental understanding of how T-cells recognize antigens requires looking at the right place, that is, within the immunological synapse formed between the T-cell and the APC. Here, molecular binding kinetics are not only determined by the inherent biochemical properties of the interaction partners involved but depend to a large extent on cellular parameters, which include cellular forces, membrane architecture and lateral interactions between membrane proteins as well as synapse-specific geometrical constraints 3. Biochemical approaches are limited in resolving power as they necessitate the disruption of at least one of the synaptic membranes involved. For this reason a FRET-based imaging methodology was developed to monitor binding of the TCR to antigenic pMHCs 2. Here T-cells are decorated with a recombinant and site-specifically labeled TCR&#946;-reactive single chain antibody fragment (scFV) and confronted with planar glass-supported lipid bilayers (SLBs), which harbor MHC class II molecules loaded with a fluorescently labeled antigenic peptide, costimulatory molecules and adhesion proteins. Synaptic binding between dye-labeled TCR and dye-labeled pMHC results in FRET, which can be monitored on a bulk and single molecule level by Total Internal Reflection Fluorescence (TIRF) microscopy.
In this article it is explained in detail how to utilize SLBs for assaying T-cell synapses, verify their integrity through a functional T-cell calcium-flux assay, conduct FRET measurements in bulk and with single molecule sensitivity, and analyze the acquired data. Recommendations are offered to producing properly conformed proteins required for bilayer functionalization. For more specific information regarding bilayer formation and setup of a suitable TIRF microscope please refer to an additional public access JoVE publication published back to back 4.
Nature of SLBs
Functionalizable SLBs can be readily generated from unilamellar vesicles (SUVs) containing the two lipids 1-palmitoyl-2-oleoyl-sn-gylcero-3-phosphocholine (short: POPC, 90-99%) and 1,2-dioleoyl-sn-glycero-3-{[N(5-amino-1-carboxypentyl) iminodiacetic acid] succinyl} (short: DGS NTA-Ni, 1-10%). SUVs spread on clean glass slides to form a contiguous planar bilayer 4. DGS-NTA-Ni serves to anchor polyhistidine-tagged proteins via polyhistidine-mediated complex-formation with the synthetic NTA-Ni-containing head group (Figure 1A). For stable association one typically replaces the native transmembrane domain and cytoplasmic tail of the adhesion protein ICAM-1 and the costimulatory molecule B7-1 with one tag containing twelve histidines (ICAM-1-12H, B7-1 -12H) (Figure 1B). The peptide-loaded class II molecule I-Ek contains two membrane-embedded (&#945; and &#946;) polypeptide chains. The transmembrane/cytoplasmic domains of both chains have to be replaced with a tag containing six histidines each (I-Ek&#945;6H &#946;6H or I-Ek-2x6H). As an alternative, extending the &#945;-chain with twelve histidines and leaving the extracellular domain of the &#946;-chain untagged (giving rise to I-Ek&#945;12H &#946;0H or I-Ek-12H) gives rise to satisfactory results (Figure 1B).
Site-specific labeling of pMHCs
It is important to label the pMHC stoichiometrically and site-specifically in order to be able to convert measured FRET yields into meaningful equilibrium binding constants. This can be achieved by chemical labeling of a synthetic peptide that is loaded into the peptide-binding cleft of recombinant histidine-tagged MHC class II molecules 2,5. The peptide includes all residues of the T-cell epitope as well as a short C-terminal linker (GGS) followed by cysteine (e.g. in the moth cytochrome c (MCC) peptide ANERADLIAYLKQATK-GGSC, the linker is marked in bold). This cysteine is used to label the peptide stoichiometrically with the use of maleimide-dye derivatives. At this point extra care should be devoted to verifying quantitative dye-coupling to the cysteine-containing peptide. HPLC-purification of the peptide-dye adduct is recommended and has to be followed by electrospray ionization mass spectroscopy. Any recorded masses corresponding to the peptide educt (without dye) reflect incomplete labeling. If this is true, the HPLC-purified peptide should be subjected to consecutive rounds of dye-labeling until labeling is deemed quantitative. Note that MALDI-TOF mass spectroscopy should be avoided as this method involves laser radiation for sample ionization. This treatment disintegrates the attached sensitive fluorophores before peptide mass is read out and thus underrepresents degrees of dye-conjugation.
Indirect yet site-specific labeling of cell-bound TCRs with the use of monovalent single chain FV fragments
It is still challenging to attach dyes to cell surface-associated proteins of living cells in a site-specific manner. To overcome this hurdle for surface-exposed TCRs, a monovalent single chain version (scFV) from the genes of the TCR&#946; -reactive monoclonal antibody H57-197 2has been constructed. The crystal structure of this antibody in complex with the TCR allows to rationally design a version, in which a serine residue in close proximity to the C-terminus of the MHC associated peptide (where the corresponding FRET partner dye is attached) is substituted for a cysteine residue. This mutant cysteine then serves as an acceptor for dye conjugation (Figure 2).
Methodologies to record FRET
Bulk FRET values are best suited to verify the relationship between chosen inter-dye distances and FRET efficiencies measured in this TCR-pMHC binding system 2. In addition, bulk FRET measurements reveal qualitative and quantitative differences in synaptic TCR-pMHC affinities (see below and protocol section 3.2). Various approaches for quantifying FRET efficiencies have been introduced in the literature 6. In this article FRET is recorded via
(a) donor recovery after acceptor bleaching, and via
(b) sensitized FRET acceptor emission.
The first method (a) requires the use of a FRET acceptor that can be easily photobleached, and a donor, which is rather photostable. In addition it is important to ensure that the photobleached acceptor is no longer capable of quenching the donor fluorescence. As the same detection channel (donor) is used for quantification, no correction factors and no chromatic aberrations have to be considered, which renders this methodology simple and reliable. However, quantitative measurements cannot be repeated on the same specimen spot and changes in FRET cannot be recorded over time. To avoid effects caused by molecular diffusion or cellular motility a fast bleaching step should be aimed for, which minimizes the time passing between the first FRET donor (before acceptor bleaching) and the second FRET donor image acquisition (after FRET acceptor bleaching). It is recommend to employ a powerful laser light source of the FRET acceptor excitation wavelength in order to minimize illumination and bleaching times.
In contrast, in the approach of sensitized FRET emission measurement (b) the FRET donor is excited and the emission of the FRET acceptor is observed in the FRET acceptor channel. Changes in FRET acceptor signal can be recorded over time but emission of the FRET donor into the red-shifted acceptor channel (termed bleedthrough) and FRET acceptor cross-excitation via donor excitation have to be accurately determined and subtracted from the recorded FRET acceptor channel. For this the corresponding FRET donor and FRET acceptor images have to be spatially aligned.
Detection of single molecule (sm) FRET events
With the use of lasers as excitation source, a sensitive camera and noise-attenuated TIRF microscopy the fluorescence of single fluorophores can be easily traced over time. Similar is true for the detection of intermolecular smFRET events. However, complications may be caused by FRET donor bleedthrough and cross-excitation of the FRET acceptor, and thus great care has to be taken when adjusting the fluorophore densities in the smFRET experiment.
In the protocol provided below (protocol section 4) the TCR was chosen as FRET donor in high abundance and pMHC as FRET acceptor in low abundance. To attenuate FRET donor bleedthrough sufficiently, decorate 10-30% of the TCRs with fluorescent scFV and 90-70% of the TCRs with non-fluorescent scFV. Here the FRET acceptor channel was chosen as single molecule channel because it is confocal with the single molecule FRET channel. This helps to align smFRET events with single molecule FRET acceptors, which is the basis of smFRET validation.
Extracting synaptic off-rates through smFRET measurements
Photobleaching of both FRET donor and FRET acceptor have to be accounted for when extracting the half-life of interactions from single molecule FRET traces. The number of observable FRET-signals at the beginning of their appearance as single donor-acceptor pair N(0) is reduced over time by both unbinding of the receptor-ligand complex and photobleaching. The number of surviving complexes at a given time N(t) can be mathematically expressed as follows:
<img alt="Equation 1" src="/files/ftp_upload/53157/53157eq1.jpg" />
In the photobleaching term exp(-t/&#964;bleach) the time t is described by the product of the number of observations n and the illumination time till because of the non-continuous, discrete observation mode (i.e., bleaching only occurs during illumination). Within the kinetic term exp-(t/&#964;off) the time t is the product of the number of observations n and the time tlag for a single FRET observation (i.e., kinetic unbinding happens continuously). Equation 1 can be expressed as:
<img alt="Equation 2" src="/files/ftp_upload/53157/53157eq2.jpg" />
The term &#964;bleach/&#964;ill describes the number of observations until bleaching occurs and is defined as the expectation value &#60;nbleach&#62; of its exponential function. Equation 2 can be simplified as follows:
<img alt="Equation 3" src="/files/ftp_upload/53157/53157eq3.jpg" />
The expectation value &#60;n(tlag)&#62; of the number of frames N(t) with observable FRET-events after time t is directly determined from the experiment. It depends on the settable time between observations (tlag) chosen in the experiment and the unknown values for &#964;off (the inverse of the off-rate koff ) and &#60;nbleach&#62;, the expectation value of number of observations before bleaching occurs.
Thus, calculation of the expectation value &#60;n(tlag)&#62; for at least two values of tlag allows the experimental determination of &#60;nbleach&#62; and &#964;off .
Extracting synaptic 2D-KD values through FRET-based measurements
Measuring TCR occupancy a, i.e., the ratio between bound TCRs and total TCRs, is central to determining synaptic 2D-KD values. According to equation 4 this term is directly proportional to the measured FRET yield as long as TCRs serves as FRET donors and pMHCs as FRET acceptors.
<img alt="Equation 4" src="/files/ftp_upload/53157/53157eq4.jpg" />
with a = TCR occupancy, C =conversion factor
C is a constant, which depends on the FRET system and the fluorophores used. It can be determined experimentally as shown below. a can be converted into a 2D-KD according to equation 5 when the initial density of TCR ligands prior to addition of T-cells to the bilayer is known. This is because of the high mobility of SLB-attached proteins and also because SLBs provide an almost inexhaustible reservoir of ligands 2.
<img alt="Equation 5" src="/files/ftp_upload/53157/53157eq5.jpg" />
with [pMHC initial] = initial density of pMHC prior to the addition of T-cells
With equations 4 and 5 one can now easily determine the synaptic 2D-KD between TCR and pMHC. This is most reliably done with FRET measurements based on donor recovery after acceptor bleaching (see protocol section 3.1).
However, to measure C the relationship between the FRET intensity IFRET (corrected for background, FRET donor bleedthrough and FRET acceptor cross-excitation) and TCR occupancy a has to be determined. For this, one needs to know the ratio R between the average fluorescence intensity of single TCR-associated FRET donor fluorophores (e.g. Cy3 or AF555) sm IFRET donor and the average intensity of single molecule FRET events sm IFRET. R depends on the FRET system in question, emission filters and camera used for fluorescence detection.
The TCR occupancy a can then be directly determined according to equation 6.
<img alt="Equation 6" src="/files/ftp_upload/53157/53157eq6.jpg" />
with R = sm IFRET donor / sm IFRET
R was determined as 1.45 for the H57 scFv- Cy3/pMHC-Cy5 system leads to:
a= bulk IFRET/ bulk ITCR-cy3 &#8226; 1.45
The relationship between the TCR occupancy a and the FRET yield can be determined by FRET donor recovery after acceptor bleaching. For this both parameters are plotted against one another for a number TCR microclusters as shown in Figure 4A.The slope of the linear fit indicates the conversion factor C (from equation 4).
As demonstrated in Figure 4A, C amounts for (a) the H57 scFV- Cy3/pMHC-Cy5 FRET system and (b) the applied microscope system configuration to 1.995. The TCR occupancy a can be readily deduced as follows:
TCR occupancy a = FRET yield &#8226; 1.995

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr >
			<td >LB-media</td>
			<td >Fisher Scientific</td>
			<td >10000713</td>
			<td >bacterial expression</td>
		</tr>
		<tr >
			<td >Sf900 II</td>
			<td >Life Technologies</td>
			<td >10227402</td>
			<td>insect cell media for baculo virus production</td>
		</tr>
		<tr >
			<td >Insect-XPRESS with L-glutamine (Lonza)</td>
			<td >Fisher Scientific</td>
			<td >10564038</td>
			<td>insect cell media for baculo virus expression</td>
		</tr>
		<tr >
			<td >Sf9 cells</td>
			<td>Life Technologies</td>
			<td>11496-015</td>
			<td>cells for virus production and expansion</td>
		</tr>
		<tr >
			<td >High Five Cells</td>
			<td>Life Technologies</td>
			<td>B855-02</td>
			<td>cells for potein expression</td>
		</tr>
		<tr >
			<td >LB-media</td>
			<td >Fisher Scientific</td>
			<td >10000713</td>
			<td>bacterial expression</td>
		</tr>
		<tr >
			<td >Centramate System</td>
			<td>Pall</td>
			<td></td>
			<td>protein concentartion from large volumes</td>
		</tr>
		<tr >
			<td >Centramate cassette 10kDa cutoff</td>
			<td>Pall</td>
			<td>OS010T12</td>
			<td>protein concentartion from large volumes</td>
		</tr>
		<tr >
			<td >Amicon Ultra-15 Centrifugal Filter Units</td>
			<td>EMD Millipore</td>
			<td>UFC900308</td>
			<td>protein concentartion</td>
		</tr>
		<tr >
			<td >Amicon Ultra-4 Centrifugal Filter Units</td>
			<td>EMD Millipore</td>
			<td>UFC800308</td>
			<td>protein concentartion</td>
		</tr>
		<tr >
			<td >Amicon Stirred Ultrafiltration Cell Model 200 mL</td>
			<td>EMD Millipore</td>
			<td >5123</td>
			<td>protein concentartion</td>
		</tr>
		<tr >
			<td >&#196;kta pure 25L</td>
			<td >GE Healthcare</td>
			<td>29-0182-24</td>
			<td>protein purification</td>
		</tr>
		<tr >
			<td >Superdex 200 10/300 GL</td>
			<td >GE Healthcare</td>
			<td >17-5175-01</td>
			<td>protein purification</td>
		</tr>
		<tr >
			<td >Superdex 75 10/300 GL</td>
			<td >GE Healthcare</td>
			<td >17-5174-01</td>
			<td>protein purification</td>
		</tr>
		<tr >
			<td >Mono Q 5/50GL</td>
			<td >GE Healthcare</td>
			<td >17-5166-01</td>
			<td>protein purification</td>
		</tr>
		<tr >
			<td >Ni Sepharose 6 Fast Flow</td>
			<td >GE Healthcare</td>
			<td >17-5318-01</td>
			<td>protein purification</td>
		</tr>
		<tr >
			<td >Tricorn 10/20 column</td>
			<td >GE Healthcare</td>
			<td >28-4064-13</td>
			<td>protein purification</td>
		</tr>
		<tr >
			<td >Gilson HPLC system</td>
			<td >Gilson</td>
			<td ></td>
			<td>purificationof fluorochrome-coupled peptides</td>
		</tr>
		<tr >
			<td >Pursuit XRs C18, 5 &#181;m particle size, 21.2*250mm column size</td>
			<td >Agilent</td>
			<td >A6000250X212</td>
			<td>purificationof fluorochrome-coupled peptides</td>
		</tr>
		<tr >
			<td >Pursuit XRs C18, 5 &#181;m particle size, 21.2*50 mm column size</td>
			<td >Agilent</td>
			<td >A6000050G212</td>
			<td>purificationof fluorochrome-coupled peptides</td>
		</tr>
		<tr >
			<td >Tricorn 10/20 column&#160;</td>
			<td >GE Healthcare</td>
			<td >28-4064-13</td>
			<td>protein purification</td>
		</tr>
		<tr >
			<td >Gilson HPLC system</td>
			<td >Gilson</td>
			<td ></td>
			<td>purificationof fluorochrome-coupled peptides</td>
		</tr>
		<tr >
			<td >Pursuit XRs C18, 5 &#181;m particle size, 21.2*250mm column size</td>
			<td >Agilent</td>
			<td >A6000250X212</td>
			<td>purificationof fluorochrome-coupled peptides</td>
		</tr>
		<tr >
			<td >Pursuit XRs C18, 5 &#181;m particle size, 21.2*50 mm column size</td>
			<td >Agilent</td>
			<td >A6000050G212</td>
			<td>purificationof fluorochrome-coupled peptides</td>
		</tr>
		<tr >
			<td >Cy3 maleimide</td>
			<td >GE Healthcare</td>
			<td>PA23031</td>
			<td>site-specific protein labeling via mutant unpaired cysteines</td>
		</tr>
		<tr >
			<td >Cy5 maleimide</td>
			<td >GE Healthcare</td>
			<td>PA25031</td>
			<td>site-specific protein labeling via mutant unpaired cysteines</td>
		</tr>
		<tr >
			<td >Alexa Fluor 555 C2 Maleimide</td>
			<td >Life Technologies</td>
			<td>A-20346</td>
			<td>site-specific protein labeling via mutant unpaired cysteines</td>
		</tr>
		<tr >
			<td >Alexa Fluor 647 C2 Maleimide</td>
			<td >Life Technologies</td>
			<td >A-20347</td>
			<td>site-specific protein labeling via mutant unpaired cysteines</td>
		</tr>
		<tr >
			<td >Fura-2, AM, cell permeant</td>
			<td >Life Technologies</td>
			<td >F-1221</td>
			<td>calcium-sensitive dye for cell labeling</td>
		</tr>
		<tr >
			<td >dimethyl sulfoxide</td>
			<td >Sigma Aldrich</td>
			<td >151874</td>
			<td>for dissolving fura-2 am</td>
		</tr>
		<tr >
			<td >Hank&#39;s Balanced Salt Solution plus calcium/magnesium</td>
			<td >Fisher Scientific</td>
			<td >10225362</td>
			<td>imaging buffer</td>
		</tr>
		<tr >
			<td >PBS</td>
			<td >Life Technologies</td>
			<td >14190-136</td>
			<td></td>
		</tr>
		<tr >
			<td >Bovine Serum Albumin lyophilized powder</td>
			<td >Sigma Aldrich</td>
			<td >A2153</td>
			<td>supplement for imaging buffer</td>
		</tr>
		<tr >
			<td >14-4-4S antibody</td>
			<td >affimetrix eBioscience</td>
			<td >14-5980-81</td>
			<td>blocking antibody for H2-I-Ek (recognized by the 5c.c7, 2B4 and AND TCR)</td>
		</tr>
		<tr >
			<td >5 ml polypropylene round-bottom tube</td>
			<td >Becton Dickinson</td>
			<td >FALCON 352063</td>
			<td></td>
		</tr>
		<tr >
			<td >0.22 &#956;m Ultrafree-MC centrifugal filter unit</td>
			<td >EMD Millipore</td>
			<td >UFC30GV0S</td>
			<td></td>
		</tr>
		<tr >
			<td >Syringe filter 0.2&#181;m</td>
			<td >Millipore</td>
			<td >GVWP04700</td>
			<td></td>
		</tr>
		<tr >
			<td >TetraSpeck Microspheres, 0.1 &#181;m, fluorescent blue/green/orange/dark red</td>
			<td >Life technologies</td>
			<td >T-7279</td>
			<td></td>
		</tr>
		<tr >
			<td >Microscope for fura-2-based calcium measurements&#160;</td>
			<td >LEICA</td>
			<td >DMI4000B</td>
			<td></td>
		</tr>
		<tr >
			<td >Microscope for (single molecule) FRET measurements</td>
			<td >LEICA/ZEISS/NIKON/OLYMPUS</td>
			<td >for details please refer to parallel JoVE contribution by Axmann et al.</td>
			<td></td>
		</tr>
		<tr >
			<td >planar supported lipid bilayers</td>
			<td ></td>
			<td >for details please refer to parallel JoVE contribution by Axmann et al.</td>
			<td></td>
		</tr>
		<tr >
			<td >RPMI 1640, with L-Glutamine</td>
			<td >Life Technologies</td>
			<td >11554416</td>
			<td>T-cell media</td>
		</tr>
		<tr >
			<td >non-essential amino acid 100X</td>
			<td >Hyclone</td>
			<td >SH30238.01</td>
			<td>T-cell media supplement</td>
		</tr>
		<tr >
			<td >penicillin/streptomycin/L-glutamine 100x</td>
			<td >Life Technologies</td>
			<td >12000226</td>
			<td>T-cell media supplement</td>
		</tr>
		<tr >
			<td >2-mercaptoethanol</td>
			<td >Sigma Aldrich</td>
			<td >M6250</td>
			<td>T-cell media supplement</td>
		</tr>
		<tr >
			<td >mouse interleukin-2 recombinant protein</td>
			<td >BPS Bioscience</td>
			<td >90185-B</td>
			<td>T-cell media supplement</td>
		</tr>
		<tr >
			<td >Research Grade Fetal Bovine Serum</td>
			<td >Hyclone</td>
			<td >SV30160.03</td>
			<td>T-cell media supplement</td>
		</tr>
		<tr >
			<td >Origin (analysis program)</td>
			<td >OrigenLab</td>
			<td >http://www.originlab.com/</td>
			<td>non-linear fitting of two parameters (tauoff, [ntlag])</td>
		</tr>
	</tbody>
</table>

measuring tcr-pmhc binding in situ using a fret-based microscopy assay

T-cells are remarkably specific and effective when recognizing antigens in the form of peptides embedded in MHC molecules (pMHC) on the surface of Antigen Presenting Cells (APCs). This is despite T-cell antigen receptors (TCRs) exerting usually a moderate affinity (&#181;M range) to antigen when binding is measured in vitro1. In view of the molecular and cellular parameters contributing to T-cell antigen sensitivity, a microscopy-based methodology has been developed as a means to monitor TCR-pMHC binding in situ, as it occurs within the synapse of a live T-cell and an artificial and functionalized glass-supported planar lipid bilayer (SLB), which mimics the cell membrane of an Antigen presenting Cell (APC) 2. Measurements are based on F&#246;rster Resonance Energy Transfer (FRET) between a blue- and red-shifted fluorescent dye attached to the TCR and the pMHC. Because the efficiency of FRET is inversely proportional to the sixth power of the inter-dye distance, one can employ FRET signals to visualize synaptic TCR-pMHC binding. The sensitive of the microscopy approach supports detection of single molecule FRET events. This allows to determine the affinity and off-rate of synaptic TCR-pMHC interactions and in turn to interpolate the on-rate of binding. Analogous assays could be applied to measure other receptor-ligand interactions in their native environment.

The recording of intracellular calcium via the fura-2 calcium dye as well as the subsequent cellular analysis to verify the stimulatory potency and hence functionality of SLBs are shown in Figure 4. As becomes evident, calcium levels rise in T-cells (expressed as the normalized fura-2 340nm/380nm ratio with baseline being 1) immediately as soon as they settle onto the stimulatory SLBs. Calcium levels return to baseline levels shortly after the addition of a antibody which blocks pMHCs from TCR engagement and which terminates T-cell activation.
Figure 6 depicts a typical experiment involving FRET donor recovery after acceptor bleaching, which is employed to measure FRET yields and which serves to calculate 2D-KD values (shown in Figure 4). Please note the increase in FRET donor intensity, which represents TCR labeled via AF555, after rapid and complete ablation of the FRET acceptor species (here: AF647 associated with pMHCs). Also evident is the strong reduction in the FRET channel, i.e., the FRET-acceptor channel under FRET donor excitation, after FRET acceptor bleaching. The barely visible remaining signal corresponds to FRET donor bleedthrough. FRET yields within individual TCR microclusters or entire synapses are calculated based on the indicated measured intensity values (Figure 6B).
Figure 7 depicts a trajectory and time lapse of a single molecule FRET event visible in two time frames. As outlined above in the introduction such behavior is caused by both decay of synaptic TCR-pMHC binding and photobleaching. To discriminate between these two contributions, the experimental acquisition time frames have to be varied in duration: while photobleaching remains a constant, changes in FRET event trajectory length are only caused by the binding kinetics. A quantitation of tracelengths, which forms the basis for the calculation of off-rates and bleaching shown in Figure 7, are provided in tables 1 to 3.
Determination of 2D-KDs requires recording of bulk FRET yields for FRET-donor labeled TCRs. With the use of the experimentally deduced constant C (Figure 4), a FRET yield measured for a TCR microcluster or an entire synapse can be converted into the TCR occupancy a, i.e., the ratio of pMHC-engaged and total TCRs (Figure 4C). With known pMHC densities present on the SLB prior to addition of the T-cell, a values can be applied to determine synaptic 2D-KD values (Figure 4D). On-rates can be calculated with the law of mass action (2D-kon = 2D-koff/2D-KD) from the determined synaptic off-rate and 2D-KD values.
<img alt="Figure 1" src="/files/ftp_upload/53157/53157fig1.jpg" /> 
Figure 1. Schematic outline of the planar glass-supported lipid bilayer (SLB) system. (A) SLBs are composed of POPC (90-99%) and the synthetic lipid DGS Ni-NTA (1-10%) and form spontaneously when clean glass surfaces are charged with small unilamellar vesicles (SUVs) consisting of the corresponding lipids. (B) Once formed, such SLBs can be functionalized with soluble polyhistidine-tagged extracellular portions derived from pMHCs, costimulatory B7-1 proteins and ICAM-1 adhesion proteins, to serve as APCs for T-cells. For more information on preparing SLBs refer to Axmann et al.4. <a href="https://www.jove.com/files/ftp_upload/53157/53157fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/53157/53157fig2.jpg" /> 
Figure 2. F&#246;rster Resonance Energy Transfer-based assay to quantitate TCR-pMHC binding in situ. (A) A composite structure of a TCR complexed with an H57 single chain fragment engaging a pMHC illustrates the FRET-based approach described herein. Note the short distance of about 41&#197; separating the two corresponding fluorophores undergoing FRET. Acceptor sites for fluorophore-maleimides are indicated in green and red. (B) The principle of detecting TCR-pMHC interactions in situ is illustrated. Only scFV-decorated TCRs and pMHCs (here I-Ek), which form specific complexes, give rise to a measurable FRET signal. <a href="https://www.jove.com/files/ftp_upload/53157/53157fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/53157/53157fig3.jpg" /> 
Figure 3. Chromatograms of the final gel filtration step giving rise to monomeric scFVs and peptide-loaded I-Ek -2x6H molecules. X-axes represent retention volume in ml, Y-axes indicate absorbance at 280 nm in arbitrary units (AU). (A) H57 scFV site-specifically labeled with Alexa Fluor 555 maleimide was subjected to S75 chromatography to separate unreacted dye from the protein (step 1.2.5). Fractions corresponding to retention interval 14 to15 ml (dashed lines) represent labeled monomeric H57 scFV. (B) I-Ek -2x6H molecules complexed with the UV-cleavable ANP-space holder peptide had been UV-irradiated, incubated with site-specifically Alex 647 maleimide-labeled peptide and finally subjected to S200 chromatography to separate the protein from free peptide (step 1.1.2.4). The interval between the dashed lines contains properly folded and monomeric pMHCs. (A, B) 0.7 ml sample was applied to the column at the start of the rum (0 ml point). Collected fractions were concentrated. The protein-to-dye ratio was determined by photospectrometry prior to dialysis against PBS/50% glycerol (for storage at -20&#176;). <a href="https://www.jove.com/files/ftp_upload/53157/53157fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" src="/files/ftp_upload/53157/53157fig4.jpg" /> 
Figure 4. Determining 2D-KDs and 2D-kons. (A) The correlation between the FRET yield as determined by donor recovery after acceptor bleaching and TCR occupancy was measured experimentally. TCR occupancy can be determined for individual TCR microclusters as explained in section 4.2. A linear fit of the data is displayed by the line, the slope of which is equal to the ratio C between TCR occupancy and the FRET yield. C is a constant specific for the FRET system and the fluorophores (here Cy3 and Cy5) employed. In this example it yielded 1.988. (B) FRET yield data were determined for individual TCR microclusters (N = 187, temperature = 24 &#176;C) through donor recovery of acceptor bleaching. Numbers below histogram bars indicate the upper limit within the interval. (C) Conversion of the data shown in (B) by multiplying measured FRET yields with the constant C determined in (A). Numbers below bars indicate the upper limit within the interval. (D) Histogram (semi-logarithmic, base = 4) depicting the distribution of 2D-KDs measured for individual TCR microclusters. The median 2D-KD is indicated in blue. Numbers below bars indicate the upper limit within the interval. (E) The histogram shown in (D) was converted into a 2D-kon -histogram (semi-logarithmic, base = 4) employing the synaptic koff for 24 &#176;C (0.41 s-1). The determined median 2D-kon value is indicated in blue. Data were originally published in Huppa et al.2 and are visualized here in a new format. <a href="https://www.jove.com/files/ftp_upload/53157/53157fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" src="/files/ftp_upload/53157/53157fig5.jpg" /> 
Figure 5. Functional validation of SLBs employed for T-cell stimulation and imaging. (A) TCR-transgenic T-cell blasts loaded with fura-2 were confronted with stimulatory SLB harboring antigenic pMHCs, ICAM-1 and B-7. Cellular fura-2 emission excited at 340 nm and 380 nm as well as DIC images were recorded. As indicated, ratio values of the emission intensities excited at 340 and 380 nm are shown in the right panel. Addition of pMHC blocking antibodies 14 min into the experimental run resulted in a decrease in intracellular calcium levels comparable to that of resting T-cells. (B) A typical temporal profile of average fura-2 ratios in T-cells contacting stimulatory SLBs is characterized by a an initial rise in intracellular calcium which is 2 to 4 times higher compared to that of non-activated T-cells or T-cells deprived of antigen after antibody-mediated blockade. Green circles indicate time points illustrated in (A). <a href="https://www.jove.com/files/ftp_upload/53157/53157fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" src="/files/ftp_upload/53157/53157fig6.jpg" /> 
Figure 6. Bulk FRET yields as measured through FRET donor recovery after FRET acceptor bleaching. (A) Shown is an example of a typical synaptic FRET-measurement. As is indicated on the left and on top a series of images was acquired with the use of a emission beam splitter giving rise to a FRET donor and a FRET acceptor channel (for more detailed information on beam splitters refer to Axmann et al4). The line shown in the left DIC image indicates the boundary of the T-cell synapse. Note the loss in intensity within the FRET acceptor channel as well is the increase in intensity in the FRET donor channel after FRET acceptor bleaching (step 4). (B) FRET efficiencies can be quantified as indicated for individual synaptic regions or for entire synapses. For inspection, images before and after FRET acceptor bleaching are shown with the use of two lookup tables (LUTs, green and physics). <a href="https://www.jove.com/files/ftp_upload/53157/53157fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" src="/files/ftp_upload/53157/53157fig7.jpg" /> 
Figure 7. Single molecule FRET events appear and disappear insingle steps and are perfectly aligned with a single FRET acceptor fluorophore.The time lapse of the single molecule FRET event is shown. Images were acquired using a back-illuminated EMCCD camera. <a href="https://www.jove.com/files/ftp_upload/53157/53157fig7large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 8" src="/files/ftp_upload/53157/53157fig8.jpg" /> 
Figure 8. Determining &#964;off = 1/ koff from measured smFRET trajectories. (A) The normalized cumulative sums of observable FRET signals (derived from H57 scFV-AF555 decorated 5c.c7 TCR transgenic T-cell blasts recognizing I-Ek/K3-AF647 at 24&#176;C) for four different time lags (42 msec, 490 msec, 1,007 msec, 1,989 msec) were plotted as a function of the total number of observations. Mono-exponential fit functions give rise to corresponding the negative inverse of the expectation values &#60;n(tlag)&#62;. (B) Expectation values were plotted against delays tlag and fitted using equation&#160;&#60;n(tlag)&#62; = &#964;off /{(&#964;off /&#60;nbleach&#62;) + tlag} to yield&#964;offand &#60;nbleach&#62;. <a href="https://www.jove.com/files/ftp_upload/53157/53157fig8large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Measuring TCR-pMHC Binding In Situ using a FRET-based Microscopy Assay at JoVE.com

Measuring TCR-pMHC Binding In Situ using a FRET-based Microscopy Assay

This manuscript describes how to conduct (single molecule) F&#246;rster Resonance Energy Transfer (FRET)- based assays to measure the binding dynamics between T-cell antigen receptor (TCR) and antigenic peptide-loaded MHC molecules as they occur within the immunological synapse of a T-cell in contact with a functionalized planar supported lipid bilayer.

Measuring TCR-pMHC Binding In Situ using a FRET-based Microscopy Assay

T-cells are remarkably specific and effective when recognizing antigens in the form of peptides embedded in MHC molecules (pMHC) on the surface of Antigen ...

measuring-tcr-pmhc-binding-in-situ-using-a-fret-based-microscopy-assay

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JoVE Journal

Bioengineering

12.2K Views.  Vienna University of Technology. This manuscript describes how to conduct (single molecule) Förster Resonance Energy Transfer (FRET)- based assays to measure the binding dynamics between T-cell antigen receptor (TCR) and antigenic peptide-loaded MHC molecules as they occur within the immunological synapse of a T-cell in contact with a functionalized planar supported lipid bilayer. 