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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualization+and+quantification+of+T+cell-mediated+cytotoxicity+using+cell-permeable+fluorogenic+caspase+substrates.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lineage+relationship+and+protective+immunity+of+memory+CD8+T+cell+subsets.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+sensitive+flow+cytometry-based+cytotoxic+T-lymphocyte+assay+through+detection+of+cleaved+caspase+3+in+target+cells.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cutting+edge:+rapid+in+vivo+killing+by+memory+CD8+T+cells.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+fluorescent+target+arrays+for+assessment+of+T+cell+responses+in+vivo.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Natural+killer+cell+education+in+mice+with+single+or+multiple+major+histocompatibility+complex+class+I+molecules.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Persistent+antigen+at+vaccination+sites+induces+tumor-specific+CD8(+)+T+cell+sequestration,+dysfunction+and+deletion.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor+regression+and+autoimmunity+after+reversal+of+a+functionally+tolerant+state+of+self-reactive+CD8++T+cells.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimized+peptide+vaccines+eliciting+extensive+CD8+T-cell+responses+with+therapeutic+antitumor+effects.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+model+for+pre-clinical+study+of+human+cancer+immunotherapy.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow+cytometry+protocols+for+surface+and+intracellular+antigen+analyses+of+neural+cell+types.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antigen+specific+killing+assay+using+CFSE+labeled+target+cells.">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

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...

Watch this Scientific Journal Video about In Vivo Assay for Detection of Antigen-specific T-cell Cytolytic Function Using a Vaccination Model at JoVE.com

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

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

in-vivo-assay-for-detection-antigen-specific-t-cell-cytolytic

Research

JoVE Journal

Immunology and Infection

9.8K Views.  University of Texas MD Anderson Cancer Center. Het doel van dit protocol is dat voor de opsporing van in vivo antigeen-specifieke doden van een cel in een lymfkliertest model.

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

Antigen Specific In Vivo Killing Assay using CFSE Labeled Target Cells

Carboxyfluorescein diacetate succinimidyl ester (CFSE) can be used to easily and quickly label a cell population of interest for in vivo investigation. This labeling has classically been used to study proliferation and migration. In the method presented here, we have shortened the timeline after adoptive transfer to look at survival and killing of epitope specific CFSE labeled target cells4-6. The level of specific killing of a CD8 + T cell clone can indicate the quality of the response, as their quantity may be misleading. Specific CD8+ T cells can become functionally exhausted over time with a decline in cytokine production and killing7,8. Also, certain CD8 + T cell clones may not kill as well as others with differing TCR specificities9. For effective Cell Mediated Immunity (CMI), antigens must be identified that produce not only adequate numbers of responding T cells, but also functionally robust responding T cells. Here we assess the percent cell specific killing of two peptide specific T cell clones in BALB/c mice.

Antigen Specific In Vivo Killing Assay using CFSE Labeled Target Cells

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.

Carboxyfluorescein diacetate succinimidyl ester (CFSE) can be used to easily and quickly label a cell population of interest for in vivo investigation.  ...

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

In vivo models of pancreatic cancer provide invaluable tools for studying disease dynamics, immune infiltration and new therapeutic strategies. The orthotopic murine model can be performed on large cohorts of immunocompetent mice simultaneously, is relatively inexpensive and preserves the cognate tissue microenvironment. The quantification of T cell infiltration and cytotoxic activity within orthotopic tumors provides a useful indicator of an antitumoral response.
This protocol describes the methodology for surgical generation of orthotopic pancreatic tumors by injection of a low number of syngeneic tumor cells resuspended in 5 &#181;L basement membrane directly into the pancreas. Mice bearing orthotopic tumors take approximately 30 days to reach endpoint, at which point tumors can be harvested and processed for characterization of tumor-infiltrating T cell activity. Rapid enzymatic digestion using collagenase and DNase allows a single-cell suspension to be extracted from tumors. The viability and cell surface markers of immune cells extracted from the tumor are preserved; therefore, it is appropriate for multiple downstream applications, including flow-assisted cell sorting of immune cells for culture or RNA extraction, flow cytometry analysis of immune cell populations. Here, we describe the ex vivo stimulation of T cell populations for intracellular cytokine quantification (IFN&#947; and TNF&#945;) and degranulation activity (CD107a) as a measure of overall cytotoxicity. Whole-tumor digests were stimulated with phorbol myristate acetate and ionomycin for 5 h, in the presence of anti-CD107a antibody in order to upregulate cytokine production and degranulation. The addition of brefeldin A and monensin for the final 4 h was performed to block extracellular transport and maximize cytokine detection. Extra- and intra-cellular staining of cells was then performed for flow cytometry analysis, where the proportion of IFN&#947;+, TNF&#945;+ and CD107a+ CD4+ and CD8+ T cells was quantified.
This method provides a starting base to perform comprehensive analysis of the tumor microenvironment.

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

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.

In vivo models of pancreatic cancer provide invaluable tools for studying disease dynamics, immune infiltration and new therapeutic strategies. The ...

Cancer Research

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

Immunogenic cell death of tumors, caused by chemotherapy or irradiation, can trigger tumor-specific T cell responses by releasing danger-associated molecular patterns and inducing the production of type I interferon. Immunotherapies, including checkpoint inhibition, primarily rely on preexisting tumor-specific T cells to unfold a therapeutic effect. Thus, synergistic therapeutic approaches that exploit immunogenic cell death as an intrinsic anti-cancer vaccine may improve their responsiveness. However, the spectrum of immunogenic factors released by cells under therapy-induced stress remains incompletely characterized, especially regarding extracellular vesicles (EVs). EVs, nano-scale membranous particles emitted from virtually all cells, are considered to facilitate intercellular communication and, in cancer, have been shown to mediate cross-priming against tumor antigens. To assess the immunogenic effect of EVs derived from tumors under various conditions, adaptable, scalable, and valid methods are sought-for. Therefore, herein a relatively easy and robust approach is presented to assess EVs' in vivo immunogenicity. The protocol is based on flow cytometry analysis of splenic T cells after in vivo immunization of mice with EVs, isolated by precipitation-based assays from tumor cell cultures under therapy or steady-state conditions. For example, this work shows that oxaliplatin exposure of B16-OVA murine melanoma cells resulted in the release of immunogenic EVs that can mediate the activation of tumor-reactive cytotoxic T cells. Hence, screening of EVs via in vivo immunization and flow cytometry identifies conditions under which immunogenic EVs can emerge. Identifying conditions of immunogenic EV release provides an essential prerequisite to testing EVs' therapeutic efficacy against cancer and exploring the underlying molecular mechanisms to ultimately unveil new insights into EVs' role in cancer immunology.

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

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.

Immunogenic cell death of tumors, caused by chemotherapy or irradiation, can trigger tumor-specific T cell responses by releasing danger-associated ...

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

The ability to monitor T cell responses in vivo is important for the development of our understanding of the immune response and the design of immunotherapies. Here we describe the use of fluorescent target array (FTA) technology, which utilizes vital dyes such as carboxyfluorescein succinimidyl ester (CFSE), violet laser excitable dyes (CellTrace Violet: CTV) and red laser excitable dyes (Cell Proliferation Dye eFluor 670: CPD) to combinatorially label mouse lymphocytes into &#62;250 discernable fluorescent cell clusters. Cell clusters within these FTAs can be pulsed with major histocompatibility (MHC) class-I and MHC class-II binding peptides and thereby act as target cells for CD8+ and CD4+ T cells, respectively. These FTA cells remain viable and fully functional, and can therefore be administered into mice to allow assessment of CD8+ T cell-mediated killing of FTA target cells and CD4+ T cell-meditated help of FTA B cell target cells in real time in vivo by flow cytometry. Since &#62;250 target cells can be assessed at once, the technique allows the monitoring of T cell responses against several antigen epitopes at several concentrations and in multiple replicates. As such, the technique can measure T cell responses at both a quantitative (e.g.&#160;the cumulative magnitude of the response) and a qualitative (e.g.&#160;functional avidity and epitope-cross reactivity of the response) level. Herein, we describe how these FTAs are constructed and give an example of how they can be applied to assess T cell responses induced by a recombinant pox virus vaccine.

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

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 &#62;250 parameters simultaneously by flow cytometry.

The ability to monitor T cell responses in vivo is important for the development of our understanding of the immune response and the design of ...

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

Traditional vaccine adjuvants, such as alum, elicit suboptimal CD8+ T cell responses. To address this major challenge in vaccine development, various nanoparticle systems have been engineered to mimic features of pathogens to improve antigen delivery to draining lymph nodes and increase antigen uptake by antigen-presenting cells, leading to new vaccine formulations optimized for induction of antigen-specific CD8+ T cell responses. In this article, we describe the synthesis of a &#8220;pathogen-mimicking&#8221; nanoparticle system, termed interbilayer-crosslinked multilamellar vesicles (ICMVs) that can serve as an effective vaccine carrier for co-delivery of subunit antigens and immunostimulatory agents and elicitation of potent cytotoxic CD8+ T lymphocyte (CTL) responses. We describe methods for characterizing hydrodynamic size and surface charge of vaccine nanoparticles with dynamic light scattering and zeta potential analyzer and present a confocal microscopy-based procedure to analyze nanoparticle-mediated antigen delivery to draining lymph nodes. Furthermore, we show a new bioluminescence whole-animal imaging technique utilizing adoptive transfer of luciferase-expressing, antigen-specific CD8+ T cells into recipient mice, followed by nanoparticle vaccination, which permits non-invasive interrogation of expansion and trafficking patterns of CTLs in real time. We also describe tetramer staining and flow cytometric analysis of peripheral blood mononuclear cells for longitudinal quantification of endogenous T cell responses in mice vaccinated with nanoparticles.

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.

Traditional vaccine adjuvants, such as alum, elicit suboptimal CD8+ T cell responses. To address this major challenge in vaccine development, various ...

A Suction Blister Protocol to Study Human T-cell Recall Responses In Vivo

Cutaneous antigen-recall models allow for studies of human memory responses in vivo. When combined with skin suction blister (SB) induction, this model offers accessibility to rare populations of antigen-specific T-cells representative of the cellular memory response as well as the cytokine microenvironment in situ.
This report describes the practical procedure of a cutaneous recall, an SB induction, and a harvest of antigen-specific T-cells. To exemplify the method, the tuberculin skin test is used for antigenic recall in individuals who, prior to this study, underwent a Bacillus Calmette-Gu&#233;rin vaccination against an infection with Mycobacterium tuberculosis. Finally, examples of multiplex and flow cytometric analyses of SB specimens are provided, illustrating high fractions of antigen-specific polyfunctional CD4+ T-cells available by this sampling method compared with cells isolated from the blood.
The method described here is safe and minimally invasive, provides a unique opportunity to study both innate and adaptive immune responses in vivo, and may be beneficial to a broad community of researchers working with cell-mediated immunity and human memory responses, in the context of vaccine development.

A Suction Blister Protocol to Study Human T-cell Recall Responses In Vivo

Here, we provide a demonstration of the suction blister cutaneous recall model. The model allows a simple access to study human in vivo adaptive immune responses, for instance in the context of vaccine development.

Cutaneous antigen-recall models allow for studies of human memory responses in vivo. When combined with skin suction blister (SB) induction, this model ...

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

Upon viral infection, antigen-specific CD8+ cytotoxic T cells (CTLs) arise and contribute to the elimination of infected cells to prevent the spread of pathogens. Therefore, the frequency of antigen-specific CTLs is indicative of the strength of the T cell response against a specific antigen. Such analysis is important in basic immunology, vaccine development, cancer immunobiology and the adaptive immunology. In the vaccine field, the CTL response directed against components of a viral vector co-determines how effective the generation of antigen-specific cells against the antigen of interest (i.e., transgene) is. Antigen-specific CTLs can either be detected by stimulation with specific peptides followed by intracellular cytokine staining or by the direct staining of antigen-specific T cell receptors (TCRs) and analysis by flow cytometry. The first method is rather time-consuming since it requires sacrificing of animals to isolate cells from organs. Also, it requires isolation of blood from small animals which is difficult to perform. The latter method is rather fast, can be easily done with small amounts of blood and is not dependent on specific effector functions, such as cytolytic activity. MHC tetramers are an ideal tool to detect antigen-specific TCRs.
Here, we describe a protocol to simultaneously detect antigen-specific CTLs for the immunodominant peptides of the viral vector VSV-GP (LCMV-GP, VSV-NP) and transgenes (OVA, HPV 16 E7, eGFP) by MHC I tetramer staining and flow cytometry. Staining is possible either directly from blood or from single cell suspensions of organs, such as spleen. Blood or single cell suspensions of organs are incubated with tetramers. After staining with antibodies against CD3 and CD8, antigen-specific CTLs are quantified by flow cytometry. Optionally, antibodies against CD43, CD44, CD62L or others can be included to determine the activation status of antigen-specific CD8+T cells and to discriminate between na&#239;ve and effector cells.

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

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).

Upon viral infection, antigen-specific CD8+ cytotoxic T cells (CTLs) arise and contribute to the elimination of infected cells to prevent the spread of ...

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

Carboxyfluorescein succinimidyl ester (CFSE)-based in vivo cytotoxicity assays enable sensitive and accurate quantitation of CD8+ cytolytic T lymphocyte (CTL) responses elicited against tumor- and pathogen-derived peptides. They offer several advantages over traditional killing assays. First, they permit the monitoring of CTL-mediated cytotoxicity within architecturally intact secondary lymphoid organs, typically in the spleen. Second, they allow for mechanistic studies during the priming, effector and recall phases of CTL responses. Third, they provide useful platforms for vaccine/drug efficacy testing in a truly in vivo setting. Here, we provide an optimized protocol for the examination of concomitant CTL responses against more than one peptide epitope of a model tumor antigen (Ag), namely, simian virus 40 (SV40)-encoded large T Ag (T Ag). Like most other clinically relevant tumor proteins, T Ag harbors many potentially immunogenic peptides. However, only four such peptides induce detectable CTL responses in C57BL/6 mice. These responses are consistently arranged in a hierarchical order based on their magnitude, which forms the basis for TCD8 &#8220;immunodominance&#8221; in this powerful system. Accordingly, the bulk of the T Ag-specific TCD8 response is focused against a single immunodominant epitope while the other three epitopes are recognized and responded to only weakly. Immunodominance compromises the breadth of antitumor TCD8 responses and is, as such, considered by many as an impediment to successful vaccination against cancer. Therefore, it is important to understand the cellular and molecular factors and mechanisms that dictate or shape TCD8 immunodominance. The protocol we describe here is tailored to the investigation of this phenomenon in the T Ag immunization model, but can be readily modified and extended to similar studies in other tumor models. We provide examples of how the impact of experimental immunotherapeutic interventions can be measured using in vivo cytotoxicity assays.

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

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.

Carboxyfluorescein succinimidyl ester (CFSE)-based in vivo cytotoxicity assays enable sensitive and accurate quantitation of CD8+ cytolytic T lymphocyte ...

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

The assessment of modern sub-unit vaccines reveals that the generation of neutralizing antibodies is important but not sufficient for adjuvant selection. Therefore, adjuvants with both humoral and cellular immuno-stimulatory capabilities that are able to promote cytotoxic T lymphocytes (CTL) responses are urgently needed. Thus, faithful monitoring of adjuvant candidates that induce cross-priming and subsequently enhance CTL generation represents a crucial step in vaccine development. In here we present an application for a method that uses SIINFEKL-specific (OT-I) T cells to monitor the cross-presentation of the model antigen ovalbumin (OVA) in vivo in the presence of different adjuvant candidates. This method represents a rapid test to select adjuvants with the best cross-priming capabilities. The proliferation of CD8+ T cells is the most valuable indication of cross-priming and it is also regarded as a correlate of adjuvant-induced cross-presentation. This feature can be evaluated in different immune organs like lymph nodes and spleen. The extent of the CTL generation can also be monitored, thereby giving insights on the nature of a local (draining lymph node mainly) or a systemic response (distant lymph nodes and/or spleen). This technique further allows multiple modifications for testing drugs that can inhibit specific cross-presentation pathways and also offers the possibility to be used in different strains of conventional and genetically modified mice. In summary, the application that we present here will be useful for vaccine laboratories in industry or academia that develop or modify chemical adjuvants for vaccine research and development.

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

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.

The assessment of modern sub-unit vaccines reveals that the generation of neutralizing antibodies is important but not sufficient for adjuvant selection. ...

Medicine

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

Source: Lirussi, D. et al., <a href="https://app.jove.com/v/57401">Rapid In Vivo&#160;Assessment of Adjuvant&#39;s Cytotoxic T Lymphocytes Generation Capabilities for Vaccine Development.</a> J. Vis. Exp. (2018)
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.

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
All mice ...

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

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