A technique combining in situ tetramer staining and in situ hybridization (ISTH) enables visualization, mapping and analysis of the spatial proximity of virus-specific CD8+ T cells to their virus-infected targets, and determination of the quantitative relationships between these immune effectors and targets to infection outcomes.
This method was used in the research reported in Li et al., Science 323, 1726-1729 (2009).
Combined in situ tetramer staining and in situ hybridization (ISTH) procedures
ISTH procedures can be divided into 4 steps: 1) IST staining of antigen-specific CD8+ T cells in tissues; 2) ISH to detect virus-infected-RNA+cells; 3) confocal microscopic imaging of IST-stained cells and viral-RNA+ cells; 4) image analysis, including construction of image montages and mapping of tetramer+ and virus RNA+ cells.
1. IST staining of antigen-specific CD8+ T cells in tissue sections.
2. Detect virus-infected-RNA+ cells by in situ hybridization.
This in situ hybridization protocol was modified from our previously published protocols4,5 as follows:
3. Acquire confocal microscope images.
4. Image analyses: montage image reconstruction, cell centroid calculation and Spatial analysis.
Spatial relationships between red tetramer+ and blue SIV RNA+ cells are captured by copying and pasting their respective plots from the Excel files into a Photoshop document. After decreasing the opacity of a layer to align and scale the grids so that they coincide, color-selecting and copying and pasting into a new layer the positions of the blue RNA+ cells, and then discarding the layer used to align the grids, the positions of the tetramer+ and viral RNA+ cells will be revealed in the flattened image.
5. Additional Notes
If cutting infectious tissue:
Work in a BSL2.5 lab. Take special precautions with razor blades. Put the razor into the vibratome blade mount with a forceps. If you can’t put the blade in with a forceps, then put it in prior to adding tissue to the bath, and don’t replace the blade. When finished cutting, remove the blade with a forceps. Spray blade with 70% EtOH or DMQ prior to removal. As always, dispose of blades in a sharps container. Change outer gloves or rinse in disinfectant after each contact with tissue or contaminated PBS in tank. When finished, if working with infectious material, add DMQ disinfectant to PBS in tank and let sit a couple of minutes prior to removing. Dispose of decontaminated PBS down the drain. Then rinse the tank with water to remove salt and disinfectant. Clean workspace and utensils with DMQ. Rinse utensils with water.
To Make tetramers from biotinylated MHC monomers:
Obtain biotinylated MHC class I molecules such as HLA-A *0201/B2m/peptide molecules produced with either Gag (SLYNTVATL), Pol (ILKEPVHGV), or irrelevant MART peptide (ELAGIGILTV) peptides from Beckman Coulter. Biotinylated HLA-A *0301/B2m/peptide molecules produced with either Gag (KIRLRPGGK) or Nef (QVPLRPMTYK) at the NIAID tetramer facility. Tetramers are generated by adding 6 aliquots of FITC labeled ExtraAvidin (Sigma) to biotinylated Mamu A*01/B2m/peptide monomers over the course of 8 hours to a final molar ratio of 4.5:1. The rationale behind adding the Extravidin slowly is that during the early time points there is an overabundance of monomer which assures that all of the Extravidin added gets all four biotin sites bound. Later in the reaction, this process is less efficient because there is less monomer left and more of a chance that ExtrAvidin will not have all 4 sites bound. If added all of the ExtrAvidin at once the reaction would be less efficient and more Extravidin molecules would have only 2 or 3 monomers attached.
Since virus RNA is used as a marker for virus-infected cells in this report, all the reagents before hybridization should be RNAase free. The in situ hybridization procedure described here has been modified to preserve the fluorescent signal from in situ tetramer staining. Since the radioautographic signal from virus-infected-cells detected by in situ hybridization is at a depth of about one-cell-diameter, the thick sections used for in situ tetramer staining have to be recut into 5-8-micron-thick sections.
This novel technique of combining in situ tetramers and in situ hybridization (ISTH) described here enables simultaneous visualization, mapping and analysis of the spatial relationship of virus-specific CD8+ T cells and virus-infected target cells. This method can be applied to evaluate CD8+ T cell based vaccine and in other non-virus pathogen and host interactions. The image analysis method described here can also be adapted to study the spatial relationships of any two-cell populations interacting in situ, e.g. we recently used the centroid mapping procedure in studying of HIV-1 sexual transmission in the nonhuman primate model of simian immune deficiency virus infection of rhesus macaques6
The authors have nothing to disclose.
We thank J. Sedgewick for assistance in setting up the confocal microscope to capture images of silver grains; Supported in part by NIH research grants AI48484 (A.T.H.), AI20048 (R.A.), and AI066314 (C.J.M.); National Center for Research Resources grant. RR00169 (CNPRC, University of California, Davis).
<p class="jove_step">Solutions and Reagents:</p> | |||
<ul> <li>PBS-H Phosphate buffered saline containing heparin (100ug/ml or 18.7U/ml) to inhibit RNASes</li> <li>RPMI tissue culture media containing heparin (100ug/ml or 18.7U/ml) to inhibit RNASes</li> <li>4% low melt agarose in PBS</li> <li>PBS with 2% normal goat serum (or serum from species in which the fluorophore conjugated antibodies were made).</li> <li>PBS with 2% normal goat serum and 0.3% triton X-100</li> <li>MHC class I tetramers conjugated to FITC</li> <li>Anti-FITC antibodies, e.g. BioDesign rabbit anti-FITC</li> <li>Fluorophore conjugated anti-rabbit IgG that has been highly cross adsorbed to other species IgG for use with multiple labeling, e.g. goat-anti-rabbit-Cy3</li> <li>Fluorophore conjugated anti-mouse IgG that has been highly cross adsorbed to other species IgG for use with multiple labeling</li> <li>Fresh PBS buffered 4% paraformaldehyde</li> <li>0.01M Urea</li> <li>Glycerol/gelatin containing 4mg/ml n-propyl gallate</li> </ul> | |||
<p class="jove_step">Special equipment</p> | |||
<ul> <li>Vibratome</li> <li>Scalpel</li> <li>Razor blades</li> <li>Surgical scissors</li> <li>Loctite Quick Set Instant Adhesive (product # 46551)</li> <li>Forceps</li> <li>#2 camel hair paint brushes. Can trim with razor blade to desired thickness</li> <li>24-well flat bottomed tissue culture plates e.g. Falcon catalog #353226</li> <li>Tissue chambers</li> <li>Tin foil</li> <li>Cardboard slide folder e.g. Fisher catalog#12-587-10</li> <li>Confocal Microscope</li> </ul> | |||
<p class="jove_step">IST Reagents Quick Reference</p> | |||
<p class="jove_step">PBS-H (phosphate buffered saline with heparin)</p> | |||
<ul> <li>450 ml 1X PBS</li> <li>50 ml 1X PBS + 10X heparin</li> </ul> | |||
<p class="jove_step">1x PBS + 10X heparin</p> | |||
<ul> <li>500 ml 1X PBS </li> <li>500 mg heparin powder (found on dry chemical storage shelf) </li> </ul> | |||
<p class="jove_step">PBS-H/ 2% NGS (normal goat serum)</p> | |||
<ul> <li>49 ml PBS-H in Falcon tube </li> <li>1 ml NGS (found in 1 ml aliquots in -20 freezer) </li> </ul> | |||
<p class="jove_step">PBS-H/ 2% NGS/ 0.3% Triton X-100</p> | |||
<ul> <li>49 ml PBS-H/ 0.3% Triton X-100 in Falcon tube </li> <li>1 ml NGS (found in 1 ml aliquots in -20 freezer) </li> </ul> | |||
<p class="jove_step">PBS-H/ 0.3% Triton X-100</p> | |||
<ul> <li>500 ml PBS-H </li> <li>1.5 ml Triton X-100 (this is a thick liquid found on dry chemical storage shelves) </li> </ul> | |||
<p class="jove_step">4% LMP Agarose</p> | |||
<ul> <li>2 g LMP agarose powder </li> <li>50 ml PBS </li> <li>Shake until mixed, microwave until powder dissolves. </li> </ul> | |||
<p class="jove_step">GG-NPG (glycerol gelatin with n-propyl galate</p> | |||
<ul> <li>Place a bottle of glycerol gelatin in 50 degree water bath to melt</li> <li>Add 0.06 g of propyl galate (found on Terri's shelf), shake well</li> <li>Keep in water bath, shaking occasionally until dissolved.</li> <li>Aliquot into 1 ml tubes (brown tubes)</li> </ul> | |||
<p class="jove_step">4% paraformaldehyde</p> | |||
<ul> <li>4 g paraformaldehyde powder (found on dry chemical storage shelves) </li> <li>Put about 10 ml PBS-H in a graduated cylinder, add pf powder </li> <li>Add some water until volume reaches about 70 ml </li> <li>Add one dropper of NaOH. </li> <li>Stir until dissolved (usually about 20-30 minutes) </li> <li>Add HCl until pH reaches between 7-8. </li> <li>Bring volume to 100 ml with water. </li> </ul> | |||
<p class="jove_step">Urea 0.01 M</p> | |||
<ul> <li>0.3 g urea (found on dry chemical storage shelves)</li> <li>500 ml water</li> </ul> |