Name: In Vivo Assay for Detection of Antigen-specific T-cell Cytolytic Function Using a Vaccination Model (Video) | JoVE Journal
Uploaded: 2017-11-28T12:30:00.000+00:00
Duration: 7 min 5 s
Description: Learn to detect antigen-specific T-cell cytolytic function in vivo using a murine vaccination model. This rapid method enables direct assessment of cytolytic potential without requiring tumor growth or infection.

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5 minutes

This work is supported by NIH research (A1R03AI120027 (RN) and 1R21AI20012 (RN)), Institutional Research Grant (RN), start-up grant (RN), and MD Anderson CIC seed grant (RN).

All methods described here have been approved by the Institutional Animal Care and Use Committee (IACUC) of the University of Texas MD Anderson Cancer Center.
1. Preparation of Peptide for the Vaccine
<ol>
	<li>Dissolve the lyophilized peptide with phosphate-buffered saline (PBS) to the appropriate concentration and vortex for 30 s. 
	NOTE: For hgp10025-33, the final concentration is 1 mg/mL and for OVA257-264, the final concentration is 0.5 mg/mL. Reconstitute peptide prior to injection. Do not store after reconstitution.</li>
</ol>
2. Isolation of Splenocytes from Transgenic Mouse
NOTE: Cell isolation from the spleen must be performed in a sterile manner.
<ol>
	<li>Euthanize the OT-1 transgenic mouse using the approved CO2 asphyxiation method and remove the spleen. 
	NOTE: Appropriate transgenic mouse is utilized in this step specific for the peptide of choice.
	<ol>
		<li>Lay the mouse on its right side. Spray the left side of the mouse with 70% ethanol (EtOH). Pull up the skin using forceps and cut the skin using surgical scissors; the spleen will be visible within the peritoneal cavity. Gently cut open the peritoneal cavity to access the spleen. Remove the spleen using forceps.</li>
	</ol>
	</li>
	<li>Disaggregate the spleen by placing it in a 70 &#181;m filter in a petri dish with 2 mL medium A (PBS with 1% fetal bovine serum (FBS)) and smashing the spleen with the end of a plunger.
	<ol>
		<li>Collect the splenocytes from the petri dish and place in a 50 mL conical tube. Wash the petri dish with 5 mL of medium A twice to collect all cells. Add medium A up to 25 mL and centrifuge the cells for 5 min at room temperature at 475 x g.</li>
	</ol>
	</li>
	<li>Aspirate the cells and resuspend in 1 mL/per spleen red blood cell (RBC) lysis buffer. Incubate at room temperature for 5 min. Add 10 mL of medium A and centrifuge at room temperature at 475 x g. Resuspend the pellet in 10 mL of medium A and remove debris by filtering the cell suspension through a 70 &#181;m filter into a clean 50 mL conical.</li>
	<li>Count cells using trypan blue and a hemocytometer. Resuspend cells in PBS to a final concentration of 106&#160;- 1006 cells/mL. For OVA257-264 specific killing, resuspend at 106 cells/mL and for hgp10025-33 specific killing, resuspend at 1006 cells/mL.
	<ol>
		<li>Spin the remaining cells at room temperature for 5 min at 475 x g. Aspirate the supernatant and resuspend in 15 mL cold PBS to wash cells. Repeat the wash step once more. Spin the cells at room temperature for 5 min at 475 x g. Aspirate the supernatant and resuspend in cold PBS according to the final volume determined in step 2.4.</li>
		<li>Transfer single cell suspension to a 1.5 mL microcentrifuge tube and keep on ice until injection into recipient C57/BL6 mouse.</li>
	</ol>
	</li>
</ol>
3. Injection of Splenocytes from Transgenic Mice
<ol>
	<li>Place recipient C57/BL6 mouse in a restrainer with the dorsal side up. Spray injection tail base area with 70% isopropyl alcohol. Administer 100 &#181;L of single cell suspension (section 2) intravenously into the tail vein using a 27 G needle with the bevel side facing up.</li>
</ol>
4. Covax Administration
NOTE: If cells are injected in the afternoon, covax should be administered the following morning within 18 h of cell injection.
<ol>
	<li>Place the mouse in a clear anesthesia box with 1 - 3% isoflurane. After mice are fully anesthetized, transfer the mice to the nose cones attached to the manifold in the anesthesia chamber. Keep mice restrained with the dorsal side up. 
	NOTE: Anesthetization is confirmed by a brief toe pinch to verify a withdrawal response is not elicited. Federal law restricts isoflurane use on the order of a licensed veterinarian.</li>
	<li>Spray injection tail base area with 70% isopropyl alcohol. Inject 100 &#181;L of peptide solution (from section 1) subcutaneously using a 27 G needle with the syringe penetrating 4 - 5 mm into the tail base region, the needle bevel side facing up. 
	NOTE: Mice are vaccinated in one flank with subcutaneous injection at the base of the tail10.</li>
	<li>Inject 100 &#181;L anti-CD40 antibody (0.5 mg/mL stock) lateral to the vaccine injection site.</li>
	<li>Carefully apply imiquimod cream 50 mg/mouse on the vaccination sites. Rub imiquimod cream on the surface until the cream is no longer visible and is fully absorbed.</li>
	<li>Inject 100 &#181;L of 100,000 IU/mL rhIL-2 protein intraperitoneally (i.p.) at the lower abdominal region. Observe mice for 5 min after they fully recover from anesthesia. 
	NOTE: Experiment is paused at this point for 3 days.</li>
</ol>
5. Isolation of Target Splenocytes for Labeling with CFSE
NOTE: Cell isolation from the spleen must be performed in a sterile manner.
<ol>
	<li>Euthanize the C57BL/6 mice according to approved CO2 asphyxiation method. Remove spleen(s) as in step 2.1.1. Disaggregate the spleen and wash splenocytes as in steps 2.2.1 - 2.2.3. Lyse red blood cells as in steps 2.3 - 2.3.2.</li>
	<li>Remove cells for counting using a hemocytometer and calculate for a final concentration of cells at 106 cells/mL in CFSE-labeling solution (described in step 7). Spin remaining cells at room temperature for 5 min at 475 x g. Aspirate supernatant.</li>
</ol>
6. Peptide Pulsing of Target Splenocytes
NOTE: Peptide pulsing must be performed in a sterile manner.
<ol>
	<li>Resuspend cells at 106 cells/mL in complete media (RPMI 1640 media with 10% FBS, 1% L-glutamine (L-Gln), 1% penicillin/streptomycin (Pen/Strep)). Divide the cells into 2 tubes (15 mL conicals). Label each tube as pulsed or unpulsed.</li>
	<li>Add peptide to the tube labeled pulsed. For OVA257-264 pulsing, add 1 &#181;g/mL and for hgp10025-33 pulsing, add 2 &#181;g/mL. 
	&#8203;NOTE: The unpulsed cells undergo the same incubation as the pulsed cells just without the addition of the peptide.
	<ol>
		<li>Incubate cells + peptide at 37 &#176;C for 1 h.</li>
	</ol>
	</li>
	<li>Add 10 mL of complete media (RPMI 1640 media with 10% FBS, 1% L-glutamine (L-Gln), 1% penicillin/streptomycin (Pen/Strep)) to each tube to wash both pulsed and unpulsed target cells. Spin remaining cells at room temperature for 5 min at 475 x g. Aspirate supernatant.</li>
</ol>
7. Preparation of CFSE for Labeling Target Splenocytes
<ol>
	<li>Prepare CFSE-labeling solution during the cell washing in step 6.3; the pulsed cells will be labeled as CFSEhi and the unpulsed as CFSElo. Prepare 1 mL of CFSE-labeling solution for 106 cells. 
	&#8203;NOTE: CFSE is light sensitive and should be protected from light at all times.
	<ol>
		<li>Prepare CFSEhi -labeling media by adding 1 uL/mL CFSE (5 mM stock solution) for a final concentration of 5 &#181;M/mL in RPMI media 1640 with 2% FBS.</li>
		<li>Prepare CFSElo -labeling media by adding 1 uL/mL CFSE (0.5 mM stock solution) for a final concentration of 0.5 &#181;M/mL RPMI media 1640 with 2% FBS.</li>
	</ol>
	</li>
</ol>
8. Labeling of Target Splenocytes with CFSE
NOTE: CFSE labeling must be performed in a sterile manner.
<ol>
	<li>Resuspend the pulsed and unpulsed target cells (from step 6.3) at 106 cells/mL CFSE-labeling media. Resuspend the pulsed cells with prepared CFSEhi-labeling media and the unpulsed cells with prepared CFSElo-labeling media.</li>
	<li>Mix cells and CFSE-labeling media by gentle inversion or swirling. Do not mix by vortexing. Incubate cells and CFSE-labeling media at 37 &#176;C for 15 min. Remix the cells every 5 min.</li>
	<li>Add 10 mL of complete media to each cell suspension and spin cells at room temperature for 5 min at 475 x g.</li>
	<li>Aspirate supernatant and resuspend cells in 10 mL cold PBS. Spin cells at 4 &#176;C for 5 min at 475 x g.</li>
	<li>Repeat step 8.4.</li>
	<li>Count the cells and mix peptide-pulsed, CFSEhi-labeled with unpulsed, CFSElo-labeled cells at a 1:1 ratio for injection into recipient mice. Keep an aliquot of 1x 106 mixed cells to use for a baseline flow cytometry assessment (section 11). 
	NOTE: The final volume for injection is 100 &#181;L per mouse. The final cell count is 10e6 cells. Loss of volume in the syringe and needle needs to be taken into account and should be estimated at 500 &#181;L.</li>
</ol>
9. Injection of Target Cells
NOTE: Keep CFSE-labeled cells protected from light prior to and during the injection as much as possible.
<ol>
	<li>Place recipient C57BL/6 mice in a restrainer with the dorsal side up. Spray injection tail base area with 70% isopropyl alcohol. Administer 100 &#181;L of single cell suspension intravenously into the tail vein with the bevel side facing up.</li>
</ol>
10. Re-isolation of Target Cells
NOTE: The timing of this step is critical and dependent upon the CTL cytotoxicity and the strength of the antigen for stimulation. For assessment of killing an OVA257-264 pulsed target, the cells need to be harvested 4 - 6 h after injection. Since CFSE-labeled cells are light sensitive, process spleens in the dark.
<ol>
	<li>Euthanize the recipient C57BL/6 mice according to the approved CO2 asphyxiation method.</li>
	<li>Remove spleen(s) as in step 2.1.1. Disaggregate the spleen and wash splenocytes as in steps 2.2.1 - 2.2.3. Lyse red blood cells as in steps 2.3 - 2.3.2.</li>
	<li>Resuspend cells in 1 mL fluorescence activated cell sorting (FACs) buffer (1% BSA+PBS) for assessment by flow cytometry.</li>
</ol>
11. Gating Logic to Determine CTL Activity by Flow Cytometry
<ol>
	<li>Perform assessment of CTL activity using a standard flow cytometry protocol. Acquire cells using the FITC channel on a flow cytometer with a 488 nm laser for excitation11.
	<ol>
		<li>Gate on live lymphocytes using the forward scatter (FSC) vs side scatter (SSC) parameters. Subgate within the live lymphocyte gate for the total CFSE-positive population. 
		NOTE: The injected CFSE-labeled cells will make up a small subset of the total lymphocytes present in the spleen. The CSFE-positive cells should appear as two distinct populations on the log scale.</li>
		<li>Use a histogram format to determine the percentage of the unpulsed (left peak, CFSElo) and pulsed (right peak, CFSEhi) populations. Loss of the CFSEhi cells indicates antigen-specific CTL activity.</li>
	</ol>
	</li>
</ol>

<ol><li>Liu, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Visualization+and+quantification+of+T+cell-mediated+cytotoxicity+using+cell-permeable+fluorogenic+caspase+substrates%5BTitle%5D)+AND+%22Nat+Med%22%5BJournal%5D)">Visualization and quantification of T cell-mediated cytotoxicity using cell-permeable fluorogenic caspase substrates.</a> Nat Med. 8 (2), 185-189 (2002).</li>
<li>Wherry, E. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Lineage+relationship+and+protective+immunity+of+memory+CD8+T+cell+subsets%5BTitle%5D)+AND+%22Nat+Immunol%22%5BJournal%5D)">Lineage relationship and protective immunity of memory CD8 T cell subsets.</a> Nat Immunol. 4 (3), 225-234 (2003).</li>
<li>He, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+sensitive+flow+cytometry-based+cytotoxic+T-lymphocyte+assay+through+detection+of+cleaved+caspase+3+in+target+cells%5BTitle%5D)+AND+%22J+Immunol+Methods%22%5BJournal%5D)">A sensitive flow cytometry-based cytotoxic T-lymphocyte assay through detection of cleaved caspase 3 in target cells.</a> J Immunol Methods. 304 (1-2), 43-59 (2005).</li>
<li>Barber, D. L., Wherry, E. J., Ahmed, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cutting+edge%3A+rapid+in+vivo+killing+by+memory+CD8+T+cells%5BTitle%5D)+AND+%22J+Immunol%22%5BJournal%5D)">Cutting edge: rapid in vivo killing by memory CD8 T cells.</a> J Immunol. 171 (1), 27-31 (2003).</li>
<li>Quah, B. J., Wijesundara, D. K., Ranasinghe, C., Parish, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+use+of+fluorescent+target+arrays+for+assessment+of+T+cell+responses+in+vivo%5BTitle%5D)+AND+%22J+Vis+Exp%22%5BJournal%5D)">The use of fluorescent target arrays for assessment of T cell responses in vivo.</a> J Vis Exp. (88), e51627(2014).</li>
<li>Johansson, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Natural+killer+cell+education+in+mice+with+single+or+multiple+major+histocompatibility+complex+class+I+molecules%5BTitle%5D)+AND+%22J+Exp+Med%22%5BJournal%5D)">Natural killer cell education in mice with single or multiple major histocompatibility complex class I molecules.</a> J Exp Med. 201 (7), 1145-1155 (2005).</li>
<li>Hailemichael, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Persistent+antigen+at+vaccination+sites+induces+tumor-specific+CD8%28%2B%29+T+cell+sequestration%2C+dysfunction+and+deletion%5BTitle%5D)+AND+%22Nat+Med%22%5BJournal%5D)">Persistent antigen at vaccination sites induces tumor-specific CD8(+) T cell sequestration, dysfunction and deletion.</a> Nat Med. 19 (4), 465-472 (2013).</li>
<li>Overwijk, W. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Tumor+regression+and+autoimmunity+after+reversal+of+a+functionally+tolerant+state+of+self-reactive+CD8%2B+T+cells%5BTitle%5D)+AND+%22J+Exp+Med%22%5BJournal%5D)">Tumor regression and autoimmunity after reversal of a functionally tolerant state of self-reactive CD8+ T cells.</a> J Exp Med. 198 (4), 569-580 (2003).</li>
<li>Cho, H. I., Celis, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Optimized+peptide+vaccines+eliciting+extensive+CD8+T-cell+responses+with+therapeutic+antitumor+effects%5BTitle%5D)+AND+%22Cancer+Res%22%5BJournal%5D)">Optimized peptide vaccines eliciting extensive CD8 T-cell responses with therapeutic antitumor effects.</a> Cancer Res. 69 (23), 9012-9019 (2009).</li>
<li>Ya, Z., Hailemichael, Y., Overwijk, W., Restifo, N. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mouse+model+for+pre-clinical+study+of+human+cancer+immunotherapy%5BTitle%5D)+AND+%22Curr+Protoc+Immunol%22%5BJournal%5D)">Mouse model for pre-clinical study of human cancer immunotherapy.</a> Curr Protoc Immunol. 108, 21-43 (2015).</li>
<li>Menon, V., Thomas, R., Ghale, A. R., Reinhard, C., Pruszak, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Flow+cytometry+protocols+for+surface+and+intracellular+antigen+analyses+of+neural+cell+types%5BTitle%5D)+AND+%22J+Vis+Exp%22%5BJournal%5D)">Flow cytometry protocols for surface and intracellular antigen analyses of neural cell types.</a> J Vis Exp. (94), (2014).</li>
<li>Durward, M., Harms, J., Splitter, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Antigen+specific+killing+assay+using+CFSE+labeled+target+cells%5BTitle%5D)+AND+%22J+Vis+Exp%22%5BJournal%5D)">Antigen specific killing assay using CFSE labeled target cells.</a> J Vis Exp. (45), (2010).</li>
</ol>

The authors have nothing to disclose.

While this protocol is straightforward, there are a few critical steps that must be carefully performed. The covax priming following injection of the antigen-specific T-cell being tested is necessary to see any killing of the pulsed targets. While it is possible that the water-based covax vaccination creates an acute inflammatory condition, for the chronic inflammatory phase, replacing the short-lived water-based formulation with a slow-antigen release oil-based approach may produce a better outcome7</s...

Multiple protocols exist to assess the cytolytic (CTL) potential of a CD8+ or CD4+ T-cell. This assessment can be readily done in vitro under controlled conditions1,2,3. In addition, infectious disease models, such as LCMV, have classically examined CTL function through the use of differentially CFSE (5-(and 6)-Carboxyfluorescein diacetate succinimidyl ester) labeled target cells where the CFSEhi-labeled cells are pulsed with a peptide and CFSElo-labeled target cells are left unpulsed. The cells are then injected at a 1:1 ratio and assessed for loss of the CFSEhi-labeled pulsed targets by flow cytometry4. Vaccine and rejection models have also used similar strategies for assessment of in vivo killing by both CD8+ and CD4+ T cells as well as NK cells5,6. This is a powerful assay, but requires the use of infectious agents that prime the immune system prior to target injection.
This protocol, on the other hand, requires no prior infection of the host and instead utilizes a vaccination strategy to prime the immune system prior to target injection. This vaccination is comprised of a water-based formulation of peptide vaccine which requires provision of an immunostimulatory cocktail called covax7, consisting of a Toll-like receptor 7 (TLR7) agonist (imiquimod cream), an agonistic anti-CD40 antibody, and interleukin-2 (IL-2) leading to synergistic combination of immunostimulatory agents for the elicitation of peptide-specific priming and robust immune response. As such, this assay provides a quick readout of CTL function as the vaccine is administered along with the cells for assessment of function only three days prior to injection of the target cells. In addition, the covax priming is strong enough that the killing capacity of the primed antigen-specific T cell can be seen between 4 and 24 h after injection.
The major limitation of this protocol is identifying the timeline in vivo for the detection of target killing that is appropriate to both the antigen and the hypothesis being tested. Careful assessment must be performed, as variations in antigen strength as well as genetic alterations being tested in T-cells could result in differential CTL function that would require a different timing detection of target killing. In addition, while the appropriate concentration of peptide for vaccination has been optimized for human melanoma antigen glycopeptide 100 (hgp10025-33) and ovalbumin257-264 (OVA257-264)8,9, use of another antigen model that may be more appropriate to a given study would require further validation. Because of anticipated differences in a target antigens&#39; capacity to stimulate CTL effector function in combination with the covax as an adjuvant, optimization of IL-2 dose concentration and dose frequency may be essential to achieve the desired goal. Overall, this protocol allows for a quick assessment of killing function in vivo and can be adapted to any given antigen.

<table><tbody><tr><td>6 to 12 week old female C57BL/6 mice&amp;nbsp;</td><td>Charles River</td><td>027</td><td>C57 Black 6 mice</td></tr><tr><td>OT-1 6-12 week old female mice&amp;nbsp;</td><td>Jackson Labs</td><td>003831</td><td></td></tr><tr><td>hgp100&lt;sub&gt;25&amp;ndash;33&lt;/sub&gt;&amp;nbsp;</td><td>CPCScientific</td><td>834139</td><td>KVPRNQDWL</td></tr><tr><td>OVA &lt;sub&gt;257&amp;ndash;264&lt;/sub&gt;&amp;nbsp;</td><td>CPCScientific</td><td>MISC-012</td><td>SIINFEKL</td></tr><tr><td>Imiquimod cream 5%&amp;nbsp;</td><td>Fougera</td><td>51672-4145-6</td><td>Aldara Cream</td></tr><tr><td>CD40-specific mAb&amp;nbsp;</td><td>BioXcell</td><td>BE0016-2</td><td>clone FGK4.5</td></tr><tr><td>rhIL-2 protein&amp;nbsp;</td><td>Hoffman LaRoche Inc</td><td>136</td><td>Recombinant human IL-2 protein</td></tr><tr><td>70% isopropyl alcohol Prep&amp;nbsp;</td><td>Kendall</td><td>S-17474</td><td></td></tr><tr><td>PBS</td><td>Life Technologies</td><td>10010-023</td><td>Phosphate Buffered Saline</td></tr><tr><td>FBS</td><td>Life Technologies</td><td>26140-079</td><td>fetal bovine serum</td></tr><tr><td>RBC lysis buffer&amp;nbsp;</td><td>Life Technologies</td><td>A10492-01</td><td>red blood cell lysis buffer</td></tr><tr><td>RPMI 1640 Media</td><td>Life Technologies</td><td>11875119</td><td></td></tr><tr><td>L-Glutamine&amp;nbsp;</td><td>Life Technologies</td><td>25030081</td><td></td></tr><tr><td>Pen/Strep</td><td>Life Technologies</td><td>15140122</td><td>penicillin/streptomycin</td></tr><tr><td>CFSE</td><td>Life Technologies</td><td>C34554</td><td>5-(and 6)-Carboxyfluorescein diacetate succinimidyl ester</td></tr><tr><td>Bovine serum albumin (BSA)</td><td>Sigma&amp;nbsp;</td><td>A4503</td><td></td></tr><tr><td>1.5 mL MCT graduated natural&amp;nbsp;</td><td>Fisher</td><td>05-408-129</td><td>microcentrifuge tube</td></tr><tr><td>70% ethanol</td><td>Fisher</td><td>BP8201500</td><td>EtOH</td></tr><tr><td>Trypan blue solution, 0.4%&amp;nbsp;&amp;nbsp;</td><td>Life Technologies</td><td>15250-061</td><td></td></tr><tr><td>Hemocytometer</td><td>Fisher</td><td>267110</td><td></td></tr><tr><td>27 gauge needle</td><td>BD</td><td>305109</td><td></td></tr><tr><td>1 mL syringe</td><td>BD</td><td>309659</td><td></td></tr></tbody></table>

in vivo assay for detection of antigen-specific t-cell cytolytic function using a vaccination model

In Vivo Assay for Detection of Antigen-specific T-cell Cytolytic Function Using a Vaccination Model

Current methodologies for antigen-specific killing are limited to in vitro use or utilized in infectious disease models. However, there is not a protocol specifically intended to measure antigen-specific killing without an infection. This protocol is designed and describes methods to overcome these limitations by allowing for the detection of antigen-specific killing of a target cell by CD8+ T cells in vivo. This is accomplished by merging a vaccination model with a traditional CFSE-labeled target killing assay. This combination allows the researcher to assess the antigen-specific CTL potential directly and quickly as the assay is not dependent upon tumor growth or infection. In addition, the readout is based on flow cytometry and so should be readily accessible to most researchers. The major limitation of the study is identifying the timeline in vivo that is appropriate to the hypothesis being tested. Variations in antigen strength and mutations in the T cells that may result in differential cytolytic function need to be carefully assessed to determine the optimal time for cell harvest and assessment. The appropriate concentration of peptide for vaccination has been optimized for hgp10025-33 and OVA257-264, but further validation would be needed for other peptides that may be more appropriate to a given study. Overall, this protocol allows a quick assessment of killing function in vivo and can be adapted to any given antigen.

Prior to injection of CFSE-labeled target cells, the 1:1 cell mixture is run on a flow cytometer to determine the baseline frequencies of both the CFSEhi and CFSElo target cells. Figure 1A shows the gating strategy to detect changes in the CFSE populations, an initial gate is made using FSC and SSC parameters. The total CFSE-positive cells are then subgated prior to assessing changes in frequency, as this population is r...

The goal of this protocol is to allow for detection of in vivo antigen-specific killing of a target cell in a murine model.

Current methodologies for antigen-specific killing are limited to in vitro use or utilized in infectious disease models. However, there is not a protocol ...

The overall goal of this experiment is to allow for detection of in vivo antigen-specific killing of a target cell in a murine model. This method can help answer questions in immunology field, such as sufficiency and detection of antigen-specific cytolytic killing of target cells in an attacked immune system and how this could be modified by manipulation of the T-cells to enhance the function in vivo. The main advantage of this technique is that it allows the researcher to assess antigen-specific cytolytic potential directly and quickly, as the assay is not depend on tumor growth or infection.Presenting this procedures will be Cara Haymaker, and Yared Hailemichael to instruct us from Department of Melanoma Medical Oncology. After anesthetizing mice, according to the text protocol, use 70%isopropyl alcohol to spray the tail base area for injection. Using a 27 gauge needle attached to a syringe, and with the bevel side facing up, penetrate four to five millimeters into the tail base region and inject 100 microliters of previously prepared peptide solution.Then, lateral to the vaccine injection site, inject 100 microliters of anti-CD4D antibody. Carefully apply 50 milligrams per mouse of imiquimod cream on the vaccination sites. Rub the cream into the surface until the cream is no longer visible and is fully absorbed.Then inject 100 microliters of 100, 000 IUs per milliliter of recombinant human rhIL-2 protein intraperitoneally, in the lower abdominal region. After isolating splenocytes from mice and lysing the red blood cells, according to the text protocol, carry out peptide-pulsing by first resuspending cells at 10 to the sixth cells per milliliter in complete medium. Divide the cells into two 15 milliliter conical tubes and label them as pulsed or unpulsed.For OVA 257 to 264 pulsing, add one microgram per milliliter of peptide to the tube labeled pulsed. Then incubate pulsed and unpulsed cells at 37 degrees Celsius for one hour. Add 10 milliliters of complete medium to both the pulsed and unpulsed tubes to wash the target cells.Then spin the remaining cells at 475 x g and room temperature, for five minutes, before aspirating the supernatent. Prepare a CSFE high-labeling medium by adding 1 microliter per milliliter of CSFE to RPMI 1640 with 2%FBS, for a final concentration of five micromolar per millimeter. Then prepare CSFE low-labeling medium by adding one microliter per milliliter of CSFE to RPMI 1640 with 2%FBS, for a final concentration of 0.5 micromolar per milliliter.To label the target splenolytes, resuspend 10 to the sixth cells per milliliter of pulsed cells with prepared CSFE high-labeling medium, and 10 to the sixth cells per milliliter of unpulsed cells with CSFE low-labeling medium. Mix the cell suspensions by gentle inversion or swirling. Then incubate the cells at 37 degress Celsius for 15 minutes, remixing the cells every five minutes.Next, add 10 milliliters of complete medium to each cell suspension, and centrifuge the cells at 475 x g and room temperature for five minutes. Then aspirate the supernatent and use 10 milliliters of cold PBS to resuspend the cells. Spin the cells at 475 x g and 4 degrees Celsius for five minutes, before repeating the PBS wash.Count the cells and mix peptide-pulsed CSFE high-labeled with unpulsed CSFE low-labeled cells at a 1:1 ratio for injection into recipient mice. Keep an aliquot of one times 10 to the sixth mixed cells to use for a baseline flow cytometry assessment. After injecting and reisolating target cells, according to the text protocol, perform assessment of CTL activity using a standard flow cytometry protocol, acquiring cells using the FITC channel with a 488 nanometer laser for excitation.Prior to injection of CSFE-labeled target cells, the 1:1 cell mixture was run on a flow cytometer to determine the baseline frequencies of both the CSFE high and CSFE low target cells. Shown here is the gating strategy to detect changes in the CSFE populations. An initial gate was made using FSC and SSC parameters.The total CSFE positive cells were then subgated, prior to assessing changes in frequency, as this population is relatively small when compared to the unlabeled endogenous spenlocytes. An example of the relative frequency of the CSFE populations prior to injection is shown here and should be close to one to one. The necessity of the covex priming is seen in this panel, where no killing of the antigen-pulsed CSFE high-target cells was observed at 24 hours post-injection.This figure demonstrates the effective killing of the antigen-pulsed CSFE high-labeled target cells, as the peak that was observed prior to injection was almost undetected, and the ratio was dramatically shifted from 50%to 1%detection. Finally, the figure also shows the kinetics of antigen-pulsed CSFE high-label target cell killing by assessing loss of this population at both 6 and 24 hours post-injection. Once mastered, this technique can be performed in two to three days, depending upon antigen and T-cell function.While attempting this procedure, it is important to remember that CSFE labeling must be uniform, and the kinetics of CTL response must be assessed, prior to performing the assay. Following this procedure, other methods like ELISA, ELISPOT, and intracellular flow cytometry staining can also be performed to assess changes in effector function and answering other additional questions. After watching this video you should be able to have a good understanding of how to assess in vivo CTL function in the absence of an infection or tumor.

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Research

JoVE Journal

Immunology and Infection

Many infections elicit a strong CTL response, but occasionally, the quantity of responding cells does not correlate to control of the pathogen1.  One measure of CTL quality is their ability to kill specifically2.  CFSE labeling of target cells can be used to investigate this CTL response quality in vivo3,4.

Antigen Specific In Vivo Killing Assay using CFSE Labeled Target Cells

This video demonstrates a monocyte-derived dendritic cell, MoDC-based assay to measure the immunogenicity of commercially available vaccines. MoDC assays investigate dendritic cell responses to antigens, pathogens, or treatments and can provide insights into immune responses, vaccine development, and immunotherapy research.

A Vaccine-Pulsed Dendritic Cell-Based In Vitro Assay to Induce Lymphocyte Proliferation

The video demonstrates an immunological method to monitor in vivo adjuvant-induced cytotoxic T lymphocyte or CTL production. A mouse is pre-injected with genetically engineered, proliferative dye-stained donor CD8+ T lymphocytes. Subcutaneous injection of ovalbumin with adjuvants generates cytotoxic CD8+ T lymphocytes that are identified post-harvesting through immunostaining and flow cytometry.

An Immunological Technique to Monitor Adjuvant-Mediated Cytotoxic T Lymphocyte Generation

We developed an in vitro assay to investigate the role of immunoregulatory pathways in the regulation of cytokine secretion by HIV-specific CD4 T cells.

In Vitro Assay to Evaluate the Impact of Immunoregulatory Pathways on HIV-specific CD4 T Cell Effector Function

The ability to monitor T cell responses in detail in vivo is important for the development of our understanding of the immune response. Here we describe the use of fluorescent target arrays (FTAs) in an in vivo T cell assay that assesses &gt;250 parameters simultaneously by flow cytometry.

The Use of Fluorescent Target Arrays for Assessment of T Cell Responses In vivo

We describe whole-animal imaging and flow cytometry-based techniques for monitoring expansion of antigen-specific CD8+ T cells in response to immunization with nanoparticles in a murine model of vaccination.

Whole-animal Imaging and Flow Cytometric Techniques for Analysis of Antigen-specific CD8+ T Cell Responses after Nanoparticle Vaccination

We present here an application for a standard immunological technique (CFSE stained OT-I proliferation) intended to rapidly monitor adjuvant-mediated cytotoxic T lymphocyte (CTL) generation in vivo. This fast estimation of CTL capacities is useful for the development of prophylactic vaccines against intracellular pathogens as well as therapeutic cancer vaccines.

Rapid In Vivo Assessment of Adjuvant's Cytotoxic T Lymphocytes Generation Capabilities for Vaccine Development

Here, we present a protocol for the ex vivo qualitative detection of antigen-specific CD8+ T cells. Analysis is possible with single cell suspensions from organs or from small amounts of blood. A broad range of studies require the analysis of cytotoxic T cell responses (vaccination and cancer immunotherapy studies).

Simultaneous Quantification of Anti-vector and Anti-transgene-Specific CD8+ T Cells Via MHC I Tetramer Staining After Vaccination with a Viral Vector

We describe here a flow cytometry-based in vivo killing assay that enables examination of immunodominance in cytotoxic T lymphocyte (CTL) responses to a model tumor antigen. We provide examples of how this elegant assay may be employed for mechanistic studies and for drug efficacy testing.

Tailoring In Vivo Cytotoxicity Assays to Study Immunodominance in Tumor-specific CD8+ T Cell Responses

This protocol describes the surgical generation of orthotopic pancreatic tumors and the rapid digestion of freshly isolated murine pancreatic tumors. Following digestion, viable immune cell populations can be used for further downstream analysis, including ex vivo stimulation of T cells for intracellular cytokine detection by flow cytometry.

Generation of Orthotopic Pancreatic Tumors and Ex vivo Characterization of Tumor-Infiltrating T Cell Cytotoxicity

This manuscript describes how to assess in vivo immunogenicity of tumor cell-derived extracellular vesicles (EVs) using flow cytometry. EVs derived from tumors undergoing treatment-induced immunogenic cell death seem particularly relevant in tumor immunosurveillance. This protocol exemplifies the assessment of oxaliplatin-induced immunostimulatory tumor EVs but can be adapted to various settings.

In Vivo Immunogenicity Screening of Tumor-Derived Extracellular Vesicles by Flow Cytometry of Splenic T Cells

Learn to detect antigen-specific T-cell cytolytic function in vivo using a murine vaccination model. This rapid method enables direct assessment of cytolytic potential without requiring tumor growth or infection.

In Vivo Assay for Detection of Antigen-specific T-cell Cytolytic Function Using a Vaccination Model

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