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Signaling is initiated through the T Cell Receptor (TCR) when it is engaged by antigenic peptide fragments bound by Major Histocompatibility Complex (pMHC) proteins expressed on the surface of antigen presenting cells (APCs). The TCR complex is composed of the ligand binding TCRαβ heterodimer that associates non-covalently with CD3 dimers (the εδ and εγ heterodimers and the ζζ homodimer)1. Upon engagement of the receptor, the CD3 ζ chains are phosphorylated by the Src family kinase, Lck. This leads to the recruitment of the Syk family kinase, Zap70, which is then phosphorylated and activated by Lck. After that, Zap70 phosphorylates the adapter proteins LAT and SLP76, initiating the formation of the proximal signaling complex containing a large number of different signaling molecules2.
The formation of this complex eventually results in calcium and Ras-dependent transcription factor activation and the consequent initiation of a complex series of gene expression programs that give rise to T cell differentiation2. TCR signals (and the resulting state of differentiation) are modulated by many other factors, including antigen potency and crosstalk with co-stimulatory/co-inhibitory, chemokine, and cytokine receptors 3-4. Studying the spatial and temporal organization of the proximal signaling complex under various stimulation conditions is, therefore, key to understanding the TCR signaling pathway as well as its regulation by other signaling pathways.
One very useful model system to study signaling initiated by the TCR at the plasma membrane in T cells is glass-supported lipid bilayers, as described previously5-6. They can be utilized to present antigenic pMHC complexes, adhesion, and co-stimulatory molecules to T cells-serving as artificial APCs. By imaging the T cells interacting with the lipid bilayer using total internal reflection fluorescence microscopy (TIRFM), we can restrict the excitation to within 100 nm of the space between the glass and the cell surface 7-8. This allows us to image primarily the signaling events occurring at the plasma membrane. As we are interested in imaging the recruitment of signaling proteins to the TCR complex, we describe a two-camera TIRF imaging system wherein the TCR, labeled with fluorescent Fab (fragment antigen binding) fragments of the H57 antibody (purified from hybridoma H57-597, ATCC, ATCC Number:HB-218) which is specific for TCRβ, and signaling proteins, tagged with GFP, may be imaged simultaneously and in real time. This strategy is necessary due to the highly dynamic nature of both the T cells and of the signaling events that are occurring at the TCR. This imaging modality has allowed researchers to image single ligands 9-11 as well as recruitment of signaling molecules to activated receptors and is an excellent system to study biochemistry in-situ12-16.
We describe here the transfection of either naïve or in-vitro activated primary T cells with expression plasmids encoding signaling molecules tagged by GFP, using the Amaxa Mouse T Cell Nucleofector Kit. In-vitro activation of T cells using peptide-loaded splenic APCs is performed as described before7. All the T cells used in our studies express the AND TCR that recognizes MCC peptide (88-103) bound to the MHC molecule I-Ek. Transfection is carried out essentially according to the manufacturer's recommendations; however, we present some tips that promote viability of the T cells after transfection. Both viability and transfection efficiency is much higher in in-vitro activated T cells than in naïve cells.
- Pre-equilibration of the medium is very important, as cell death will result from the re-suspension of cells after transfection in medium that is either cold or has a pH other than 7.2.
- It is important to perform a titration of plasmid dose for a fixed number of cells for each construct to be used for transfection. For some larger constructs, more DNA may be required. DNA should be free of endotoxins.
- The manufacturers recommend spinning the cells at less than 90x g; in our case, this speed corresponds to 75x g. The chosen relative centrifugal force (RCF) to obtain a cell pellet is perhaps the most crucial variable, as centrifugation at low RCF precludes damage to the cell membrane.
- After electroporating the cells, it is best to immediately transfer them to the pre-equilibrated culture medium. The manufacturers recommend that the cells be kept in the nucleofector medium for no longer than 15 minutes.
- Three to four hours of incubation are generally sufficient for T cells to detectably express the GFP tagged proteins. We chose to image cells at this time point because the level of expression is low and artifacts of over-expression should be minimized. If over-expression of a particular protein is desired, however, T cells should be removed from the Amaxa medium after 4 hours. This is accomplished by again pelletting the cells at low RCF and resuspending in pre-equilibrated T cell culture medium supplemented with 50 U/mL of Interleukin 2 (IL-2) at a density of 2 million cells/mL.
Below we describe how to align the microscope for two channel simultaneous imaging. The steps involved are aligning the TIRF illumination, adjusting the laser power, and aligning the two output images using sub-resolution microbeads.
-T cells are extremely sensitive to laser radiation, and the illumination is limited to a total of 50 μW.
Once the microscope is set up, the next task is to prepare flow chambers containing glass supported lipid bilayers presenting antigen and adhesion molecules as previously described 6 and then perform TIRFM of transfected T cells interacting with the substrate. The published bilayer protocol5-6 was followed as published except that the liposomes were not incubated for 20 minutes on the coverglass after the flow chamber was assembled. Instead, HBS-BSA buffer was flowed in the flow cell immediately after assembly.
The software outputs images from the two cameras in a single frame. The output of each camera is 512 x 512 pixels; hence, the output image is 1024 x 512 pixels in size. The images first need to be split into individual frames corresponding to the two cameras. The images of sub-resolution beads from the two channels are overlaid and alignment parameters are determined. A feature specific to our system is a 1 degree rotation of one channel with respect to the other (see Figure 3). The most likely source of this rotation is the camera stands. After this rotation further relative linear transformations in X and Y need to be performed to perfectly align the beads with respect to each other. Images of beads are obtained in every experiment to determine the precise parameters to align the two channels. These linear and rotational transformations are then applied to all the images and then they are subject to segmentation algorithms and co-localization analysis.
The system described here can be adapted to study signaling downstream of any receptor at the plasma membrane. It is particularly informative in studying TCR signaling because signaling occurs in optically resolved clusters called TCR microclusters or in endosomes as recently described18. If signaling occurred in sub-resolution clusters of receptors or in un-clustered receptors additional experiments would have to be done to interpret the data. As mentioned before, the CD3 chains of the TCR complex undergo phosphorylation by Src family kinase Lck upon engagement of the TCR by peptide-MHC complexes. The phosphorylated CD3ζ chain then recruits the Syk family kinase Zap70. Visualizing the recruitment of Zap70 thus reports on the phosphorylation status of CD3ζ chain. The strength of TCR stimulation can be altered by using peptides mutated at the TCR contact residues. Such peptides are called altered peptide ligands (APLs). A representative experiment is shown in Figure 4 where the agonist peptides robustly recruits Zap70 to TCR microclusters while the lower potency peptide T102L fails to recruit Zap70 to TCR microclusters, demonstrating that the CD3ζ chain is not phosphorylated in this case. We could draw this conclusion because we had to just observe the recruitment of Zap70 to TCR clusters that were easily detected by TIRFM. An automated segmentation algorithm was used to count the number of Zap70 microclusters per cell. Also shown in the supplementary movie is the simultaneous imaging of TCR and Zap70 associated fluorescence. Note the dynamic nature of lamelipodium.
Figure 1. Image and schematic of the custom TIRF launch. Panel A shows the photograph of the TIRF launch as installed on the microscope, and Panel B is a schematic of the TIRF launch demonstrating the light path. The schematic and image showing the position of the collector lens, the beam splitter, the fiber launch with X, Y and Z adjustments, the collimating lens L1, and the focusing lens L2 installed in the dichroic cube and the TIRF objective. The inset shows the focusing lens installed in the dichroic cube.
Figure 2. Image and schematic of the two camera system. Panel A shows the photograph of the two camera system as attached to the microscope, and Panel B is a schematic of the same. The schematic and image show the position of the objective, mirror, dichroic, tube lenses TL1 and TL2, emission filters F1 and F2, the two cameras C1 and C2 on their stands with their respective X, Y and Z adjustments.
Figure 3. Use of sub-resolution beads to align the two channels. Panels A and B show overlays of sub-resolution beads before (Panel A) and after (Panel B) alignment. Panel A shows that one of the channels is rotated with respect to the other. Panels C and D are a sub section of the images in A and B. Panel E shows a two channel overlay image of cells without processing. Alignment parameters used in panel D were applied to the image shown in panel F. Notice how the lamelipodium from the two channels is perfectly aligned in panel F and not in panel E. Scale bar 4 μm for all the panels.
Figure 4. Zap70 recruitment to TCR microclusters in response to different APLs. In-vitro activated AND T cells were transfected with a plasmid encoding Zap70 fused to GFP and were incubated on glass supported Ni-NTA bilayers containing 6 molecules/μm2 of peptide loaded His-tagged-I-Ek, 100 molecules/μm2 of Alexa647 conjugated His-tagged ICAM-1 and 100 molecules/μm2 of GPI-anchored CD80. The peptides loaded are indicated in the inset. ICAM-1 refers to cells on bilayers containing 100 molecules/μm2 of Alexa-647 conjugated His-tagged ICAM-1. Dual channel simultaneous acquisition TIRF microscopy was performed in the continuous presence of Alexa546 conjugated H57 Fab fragment (non-blocking) to stain the TCR. Cells were imaged up to one hour after initial contact with the bilayer. Numbers of Zap70 clusters per cell were analyzed using an automated cluster counting software. At least 40 cells were analyzed in each case. Scale bar 2 μm for all the images.
Figure 5. Alignment of the two camera system using two different types of cameras. This panel shows the aligned overlay of sub-resolution beads imaged using the two camera system where the two cameras have different pixel sizes (Green: 6.45 μm pixel size imaged at 2x2 binning and Red: 16 μm pixel size imaged at no binning). Inset shows the magnified view of the square box in the center of the field. Scale bar 5 μm.
Supplementary Movie. Zap70 recruitment to TCR microclusters in response to agonist peptide. In-vitro activated AND T cells were transfected with a plasmid encoding Zap70 fused to GFP and were incubated on glass supported Ni-NTA bilayers containing 6 molecules/μm2 of peptide loaded His-tagged-I-Ek, 100 molecules/μm2 of Alexa647 conjugated His-tagged ICAM-1 and 100 molecules/μm2 of GPI-anchored CD80. Dual channel simultaneous acquisition TIRF microscopy was performed in the continuous presence of Alexa546 conjugated H57 Fab fragment (non-blocking) to stain the TCR. A field of transfected cells was repeatedly imaged 40 times with a time resolution of 10 seconds per frame. Scale bar 2 μm. Click here to view supplementary movie.
We describe here a system to study signaling in antigen-specific primary mouse T cells using TIRFM and glass supported lipid bilayers as artificial APCs. The technique relies on successfully expressing GFP tagged proteins in these cells. Transfecting T cells is always a challenging task. Typically, electroporation or gene delivery using retroviruses or lentiviruses are used. One technique is not superior over the other; both have their limitations and advantages. We find that electroporation has the following advantages: 1) It is not required that the cells be actively dividing as is required for retroviruses but not lentiviruses, and 2) The expression level can be controlled by varying the time the cells are incubated before imaging. The biggest drawback is that we find it very difficult to express proteins that are large in size in primary cells. We have presented a method that gives us very good cell viability after electroporation. As a result it is applicable to using it to achieve siRNA mediated gene silencing.
We also describe here a customized two channel simultaneous acquisition TIRF microscope that is chromatically corrected and does not require separate alignment for different wavelengths. These capabilities; however, are available commercially. Our system is based on principles of TIRF microscopy published by experts in the field and is cheaper than commercial alternatives. One possible criticism of our design is that we are using the same angle of incidence for the different excitation wavelengths. This would result in a different TIRF penetration depth for different excitation wavelengths. We think that these effects are small because the evanescent wave is an exponentially decaying field and the depth of the TIRF field is a linear function of wavelength. Fluorophores close to the glass surface will experience no difference in laser intensities between the two wavelengths. For fluorophores near the penetration depth, the intensity difference will be 1.3 fold for the two wavelengths 488 and 640 nm and 1.15 fold for the two wavelengths 488 and 561 nm. The same system can be used in conjunction with super resolution techniques that make use of TIRF illumination.
We have also described a two camera system which is custom built. Like the TIRF system, similar apparatuses are also commercially available. Our system offers flexibility to change the filters and dichroics, allowing its use with different combinations of wavelengths. The drawback of our system is the one degree of rotation needed to align the two images. This issue can be resolved by using camera stands that are piezo driven and offer rotational degrees of alignment. We have also successfully implemented a two camera system on this microscope, in which the cameras are different and have different pixel sizes. A system like this would be useful if one needed sensitivity in one channel and a large dynamic range in another, both of which are rarely available in the same camera. We used the Photometrics HQ-2 camera at 2x2 binning giving us an effective pixel size of 12.9 μm with the Photometrics Quant-EM camera which has a pixel size of 16 μm. A 180 mm focal length tube lens was used for the Quant-EM and a 145 mm focal length tube lens was used for the HQ-2 camera. The HQ-2 was controlled using Metamorph software and the Quant-EM was controlled via micro-manager software on a separate computer in external trigger mode. The external trigger was provided to the Quant-EM camera using a digital output of the measurement computing DAC board, which was implemented as an additional shutter in the configure illumination settings of Metamorph. The ratio of the focal lengths of the tube lenses matches the ratio of the effective pixel sizes. A representative alignment is shown in Figure 5.
All animals used in these experiments were maintained in a specific pathogen-free environment, and the experiments were approved by the National Institutes of Health Animal Care and Use Committee.
This research was supported by the Division of Intramural Research of the National Institute for Allergy and Infectious Diseases, National Institutes of Health. We are grateful to Johannes Huppa and Mark Davis for providing us with the scFv of H57 used in these studies. RV would like to thank Keir Neumann for helpful discussion during the development of this technique.