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 ...

<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>

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.

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			<td height="20" style="width:199pt;height:15.0pt" width="265">6- to 12-week old female C57BL/6 mice&#160;</td>
			<td class="xl69" style="width:110pt" width="146">Charles River</td>
			<td class="xl70" style="width:89pt" width="119">027</td>
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			<td class="xl71" height="20" style="width:199pt;height:15.0pt" width="265">OT-1 6-12-week old female mice&#160;</td>
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			<td class="xl70" style="width:89pt" width="119">003831</td>
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			<td height="24" style="height:18.0pt">hgp10025&#8211;33&#160;</td>
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			<td height="24" style="height:18.0pt">OVA 257&#8211;264&#160;</td>
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			<td height="20" style="height:15.0pt">Imiquimod cream 5%&#160;</td>
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			<td height="20" style="height:15.0pt">CD40-specific mAb&#160;</td>
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			<td>clone FGK4.5</td>
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			<td height="20" style="height:15.0pt">rhIL-2 protein&#160;</td>
			<td class="xl69">Hoffman LaRoche Inc</td>
			<td class="xl70" style="width:89pt" width="119">136</td>
			<td>Recombinant human IL-2 protein</td>
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			<td height="20" style="height:15.0pt">70% isopropyl alcohol Prep&#160;</td>
			<td class="xl69">Kendall</td>
			<td class="xl70" style="width:89pt" width="119">S-17474</td>
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			<td height="20" style="height:15.0pt">PBS</td>
			<td class="xl69">Life Technologies</td>
			<td class="xl70" style="width:89pt" width="119">10010-023</td>
			<td>Phosphate Buffered Saline</td>
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			<td class="xl71" height="20" style="width:199pt;height:15.0pt" width="265">FBS</td>
			<td class="xl70" style="width:110pt" width="146">Life Technologies</td>
			<td class="xl70" style="width:89pt" width="119">26140-079</td>
			<td>fetal bovine serum</td>
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		<tr height="20" style="height:15.0pt">
			<td class="xl71" height="20" style="width:199pt;height:15.0pt" width="265">RBC lysis buffer&#160;</td>
			<td class="xl70" style="width:110pt" width="146">Life Technologies</td>
			<td class="xl70" style="width:89pt" width="119">A10492-01</td>
			<td>red blood cell lysis buffer</td>
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		<tr height="20" style="height:15.0pt">
			<td class="xl71" height="20" style="width:199pt;height:15.0pt" width="265">RPMI 1640 Media</td>
			<td class="xl70" style="width:110pt" width="146">Life Technologies</td>
			<td class="xl70" style="width:89pt" width="119">11875119</td>
			<td></td>
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		<tr height="20" style="height:15.0pt">
			<td class="xl71" height="20" style="width:199pt;height:15.0pt" width="265">L-Glutamine&#160;</td>
			<td class="xl70" style="width:110pt" width="146">Life Technologies</td>
			<td class="xl70" style="width:89pt" width="119">25030081</td>
			<td></td>
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		<tr height="20" style="height:15.0pt">
			<td class="xl71" height="20" style="width:199pt;height:15.0pt" width="265">Pen/Strep</td>
			<td class="xl70" style="width:110pt" width="146">Life Technologies</td>
			<td class="xl70" style="width:89pt" width="119">15140122</td>
			<td>penicillin/streptomycin</td>
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		<tr height="20" style="height:15.0pt">
			<td class="xl71" height="20" style="width:199pt;height:15.0pt" width="265">CFSE</td>
			<td class="xl70" style="width:110pt" width="146">Life Technologies</td>
			<td class="xl70" style="width:89pt" width="119">C34554</td>
			<td>5-(and 6)-Carboxyfluorescein diacetate succinimidyl ester</td>
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			<td class="xl71" height="20" style="width:199pt;height:15.0pt" width="265">Bovine serum albumin (BSA)</td>
			<td class="xl70" style="width:110pt" width="146">Sigma&#160;</td>
			<td class="xl70" style="width:89pt" width="119">A4503</td>
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		<tr height="20" style="height:15.0pt">
			<td class="xl71" height="20" style="width:199pt;height:15.0pt" width="265">1.5 mL MCT graduated natural&#160;</td>
			<td class="xl70" style="width:110pt" width="146">Fisher</td>
			<td class="xl70" style="width:89pt" width="119">05-408-129</td>
			<td>microcentrifuge tube</td>
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		<tr height="20" style="height:15.0pt">
			<td class="xl71" height="20" style="width:199pt;height:15.0pt" width="265">70% ethanol</td>
			<td class="xl70" style="width:110pt" width="146">Fisher</td>
			<td class="xl73">BP8201500</td>
			<td>EtOH</td>
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		<tr height="20" style="height:15.0pt">
			<td class="xl71" height="20" style="width:199pt;height:15.0pt" width="265">Trypan blue solution, 0.4%&#160;&#160;</td>
			<td class="xl70" style="width:110pt" width="146">Life Technologies</td>
			<td class="xl70" style="width:89pt" width="119">15250-061</td>
			<td></td>
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			<td class="xl71" height="20" style="width:199pt;height:15.0pt" width="265">Hemocytometer</td>
			<td class="xl70" style="width:110pt" width="146">Fisher</td>
			<td class="xl70" style="width:89pt" width="119">267110</td>
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			<td class="xl71" height="20" style="width:199pt;height:15.0pt" width="265">27 gauge needle</td>
			<td class="xl70" style="width:110pt" width="146">BD</td>
			<td class="xl70" style="width:89pt" width="119">305109</td>
			<td></td>
		</tr>
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			<td class="xl71" height="20" style="width:199pt;height:15.0pt" width="265">1 mL syringe</td>
			<td class="xl70" style="width:110pt" width="146">BD</td>
			<td class="xl70" style="width:89pt" width="119">309659</td>
			<td></td>
		</tr>
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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 ...

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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9.7K vistas.  University of Texas MD Anderson Cancer Center. 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-spec...