This work was supported by Canadian Institutes of Health Research (CIHR) grants MOP-130465 and PJT-156295 to SMMH. JC is partially supported by a Queen Elizabeth II Graduate Scholarship in Science and Technology from the Ontario Ministry of Training, Colleges and Universities. CEM was a recipient of an Alexander Graham Bell Canada Graduate Scholarship (doctoral) from Natural Sciences and Engineering Research Council of Canada (NSERC).

The experiments described here follow animal use protocols approved by institutional entities and adhered to established national guidelines.
1. Inoculation of C57BL/6 Mice with T Ag-expressing Tumor Cells
<ol>
	<li>Grow the SV40-transformed fibrosarcoma cell line C57SV (or a similar T Ag+ adherent cell line) in Dulbecco&#8217;s modified Eagle&#8217;s medium (DMEM) with 4.5 g/L D-glucose and L-glutamine (1x) and supplemented with 1 mM sodium pyruvate and 10% heat-inactivated fetal bovine serum (FBS) in tissue culture-treated flasks at 37 &#176;C in humidified atmosphere containing 10% CO2.</li>
	<li>Once the cells become fully confluent or slightly overconfluent, gently remove and discard the medium and rinse the monolayer with pre-warmed sterile phosphate-buffered saline (PBS). 
	NOTE: Maximal T Ag expression is achieved when T Ag+ cells reach 100% confluency.</li>
	<li>Inside a biological safety cabinet, add pre-warmed trypsin-EDTA (0.25%) to cover the monolayer at room temperature until the cells are dislodged in patches. Tap the sides of the culture flask(s) several times to release the remaining adherent cells. 
	NOTE: If necessary and to expedite the trypsinization process, transfer the flask(s) into a 37 &#176;C incubator. Dislodged cells will quickly adopt a rounded shape under a light microscope. This step should last approximately 5 min.</li>
	<li>Add 5 mL of DMEM medium and dissociate clumps to prepare a single-cell suspension by pipetting the content of each flask up and down.</li>
	<li>Transfer the cell suspension through a cell strainer with 70-&#181;m pores into a tube.</li>
	<li>Spin down the tube at 400 x g for 5 min at 4 &#176;C.</li>
	<li>Discard the supernatant. Resuspend pelleted cells in 10 mL of sterile cold PBS.</li>
	<li>Repeat steps 1.6 and 1.7 twice.</li>
	<li>Count cells using a hemocytometer. Prepare a uniform suspension containing 4 x 107 cells/mL sterile PBS.</li>
	<li>Inject 500 &#181;L of the above suspension intraperitoneally (i.p.) into each adult (6-12-week-old) male or female C57BL/6 mouse.</li>
</ol>
2. Treatment Regimens
<ol>
	<li>Treatment Regimen to Examine the Contribution of nTreg Cells to TCD8 Immunodominance 
	<ol>
		<li>Four days before in vivo priming of C57BL/6 mice with C57SV cells (step 1.10), inject each animal once i.p. with 0.5 mg of a low-endotoxin, azide-free anti-CD25 monoclonal antibody (mAb) (clone PC-61.5.3), which depletes nTreg cells, or with a rat IgG1 isotype control (e.g., clone KLH/G1-2-2, clone HRPN, or clone TNP6A7).</li>
	</ol>
	</li>
	<li>Treatment Regimen to Test the In Vivo Significance of PD-1-PD-L1(2) Interactions in Shaping TCD8 Immunodominance 
	NOTE: The engagement of PD-1 by PD-L1 often, but not always, mediates the co-inhibition and/or exhaustion of Ag-specific TCD8. Therefore, treatment with anti-PD-1 can be performed in parallel with administration of anti-PD-L1 and anti-PD-L2 mAbs to reveal the exact intercellular interaction involved in a biological phenomenon.</li>
</ol>
3. Preparation of Target Splenocytes
<ol>
	<li>Euthanize sex-matched na&#239;ve C57BL/6 mice (6-12 weeks of age) that will serve as splenocyte donors by cervical dislocation.</li>
	<li>Position each mouse with its abdomen facing up inside a biological safety cabinet. Spray the skin with 70% (v/v) EtOH. Using sterile forceps and scissors, lift the skin and make a small ventral midline incision. Then, cut the skin in a cross-like fashion to expose the peritoneum.</li>
	<li>Using forceps, pull up the peritoneum in a tent-like fashion without snatching any of the internal organs. Cut the peritoneum open to expose the peritoneal cavity and gently remove the spleen.</li>
	<li>Place the spleen(s) inside a 15 mL Dounce tissue grinder containing 5 mL of sterile PBS. Apply manual pressure using the grinder&#8217;s glass plunger until the splenic tissue dissipates into a red homogenous cell suspension. 
	NOTE: Depending on the number of recipient animals per experimental group, several donor mouse spleens may be needed for target cell preparation. Up to 3 spleens can be homogenized together inside a 15 mL grinder.</li>
	<li>Transfer the homogenate into a 15 mL tube. Spin down the tube at 400 x g for 5 min at 4 &#176;C.</li>
	<li>Discard the supernatant. Resuspend pelleted cells in 4 mL of ammonium-chloride-potassium (ACK) lysing buffer for 4 min to eliminate erythrocytes. 
	NOTE: This is a time-sensitive step. Overexposing splenocytes to ACK lysing buffer will increase their fragility and render them susceptible to non-specific cell death.</li>
	<li>To each tube, add 8 mL of RPMI 1640 medium containing 10% heat-inactivated FBS, L-alanyl-L-glutamine, 0.1 mM minimum essential media (MEM) nonessential amino acids, 1 mM sodium pyruvate, 10 mM HEPES, and 1x penicillin/streptomycin, which will hereafter be referred to as complete RPMI medium (Table of Materials).</li>
	<li>Transfer the content through 70 &#181;m pores of a cell strainer into a new 15 mL tube.</li>
	<li>Spin down the tube at 400 x g for 5 min at 4 &#176;C.</li>
	<li>Discard the supernatant. Resuspend pelleted cells in 12 mL of complete RPMI.</li>
	<li>Split the splenocyte suspension into 3 equal portions (4 mL each) in 3 separate tubes.</li>
</ol>
4. Coating Target Splenocytes with Irrelevant and Cognate Peptides
<ol>
	<li>Label the tubes according to the peptides that will be used to pulse target splenocytes. Control splenocytes will be pulsed with an irrelevant peptide, and each population of cognate target splenocytes will be pulsed with a synthetic peptide corresponding to the T Ag-derived immunodominant epitope (site IV) or a subdominant T Ag epitope (site I or site II/III) (Table 1). 
	NOTE: The choice of irrelevant peptides depends on the experimental set-up and the mouse strain used in each investigation. The authors often use gB498-505 (an H-2Kb-restricted immunodominant peptide epitope of herpes simplex virus [HSV]-1) and/or GP33-41 (an H-2Db-restricted immunodominant peptide epitope of lymphocytic choriomeningitis virus ([LCMV]) in C57BL/6 mice (Table 1). These peptides are optimal choices because: (i) they are derived from pathogens not previously encountered in the mouse model described here; (ii) similar to T Ag-derived peptides, gB498-505 and GP33-41 are restricted by and binds to H-2b molecules. In &#8216;three-peak&#8217; in vivo killing assays, each of the two peaks that correspond to cognate target cells may represent splenocytes pulsed with an immunodominant or subdominant peptide. The choice of each peptide set varies according to the objectives of each experiment. See Figure 1 and Figure 2 as examples of such variation. For the remainder of this protocol, T Ag-derived sites I and IV will represent subdominant and immunodominant peptides, respectively.</li>
	<li>Pulse the content of each labeled tube with 1 &#181;M of the respective peptide for 1 h at 37 &#176;C and 5% CO2.</li>
	<li>Use a separate cell strainer (with 70-&#956;m pores) for each tube to remove clumps and debris if necessary.</li>
	<li>Spin down the tube at 400 x g for 5 min at 4 &#176;C. Discard the supernatant.</li>
	<li>Resuspend pelleted cells in 12 mL of sterile cold PBS and repeat step 4.4 once more. 
	NOTE: It is important to remove as much FBS as possible because FBS can bind CFSE in the next step.</li>
</ol>
5. Labeling target splenocytes with CFSE
<ol>
	<li>Resuspend peptide-pulsed splenocytes in 4 mL of sterile PBS.</li>
	<li>Add CFSE at 0.025 &#956;M, 0.25 &#956;M, and 2 &#956;M into the tubes containing irrelevant peptide-, site I-, and site IV-pulsed splenocytes, respectively. 
	NOTE: To achieve uniform CFSE labeling, hold each tube at a 45&#176; angle before adding CFSE to the side slightly above the cell suspension followed immediately by gentle vortexing. This will ensure the appearance of smooth histograms at the end. Batch-to-batch and age-dependent variations in CFSE intensities are not uncommon. Therefore, one may need to experiment with differential CFSE doses before deciding on optimal concentrations to be used. 
	CAUTION: CFSE is toxic at concentrations that are higher than 5 &#956;M.</li>
	<li>Place the tubes inside a 37 &#176;C incubator for 15 min and invert them once every 5 min.</li>
	<li>Add 3 mL of heat-inactivated FBS to each tube to stop the CFSE reaction. Top up the content with sterile PBS.</li>
	<li>Spin down the tube at 400 x g for 5 min at 4 &#176;C. Discard the supernatant.</li>
	<li>Resuspend pelleted cells in 12 mL of sterile PBS and repeat step 5.5.</li>
</ol>
6. Examination of Adequate/Equal CFSE Labeling of Target Splenocyte Populations
<ol>
	<li>Resuspend pelleted cells in 3 mL of PBS.</li>
	<li>Vortex the tubes gently. Transfer 10 &#956;L, each, of CFSElow, CFSEintermediate (int), and CFSEhigh cell suspensions into a 5 mL round-bottom polystyrene fluorescence-activated cell sorting (FACS) tube containing 200 &#956;L OF PBS.</li>
	<li>Interrogate cells using a flow cytometer equipped with a 488 nm laser. Draw a lymphocyte gate based on forward scatter (FSC) and side scatter (SSC) properties of the cells before acquiring 5000 events falling within the lymphocyte gate in the FL-1 channel.</li>
	<li>Within the &#8216;parent&#8217; CFSE+ population, draw additional histogram gates to identify CFSElow, CFSEint, and CFSEhigh subpopulations.</li>
	<li>Confirm equal or near-equal event numbers within the three gates. If necessary, adjust cell numbers in the &#8216;source&#8217; tubes (step 6.1) before mixing and injecting target splenocytes into na&#239;ve and primed mice in section 7.</li>
</ol>
7. Injection of CFSE-labelled Target Cells into Na&#239;ve and T-Ag-primed Recipients
<ol>
	<li>Gently vortex the source tubes. Transfer the three CFSE-labeled cell suspensions in equal ratios into a new tube.</li>
	<li>Top up the content with sterile PBS.</li>
	<li>Spin down the tube at 400 x g for 5 min at 4 &#176;C. Resuspend pelleted cells with sterile PBS.</li>
	<li>Count cells in trypan blue by a hemocytometer to ensure cellular viability of at least 95%.</li>
	<li>Adjust the volume in order to inject 1 x 107 mixed target cells/200 &#956;L PBS intravenously (i.v.), via tail vein, into each recipient C57BL/6 mouse. 
	NOTE: Store the cells on ice in between injections. Gently mix target cells prior to each injection. Record the exact time of injection for each mouse, which will determine when the animal will need to be euthanized. It is important to keep the duration of in vivo cytotoxicity consistent among all animals in the same experiment.</li>
</ol>
8. Data Acquisition
<ol>
	<li>Two or four hours after the injection of CFSE-labeled target cells, euthanize the recipient mice by cervical dislocation. 
	NOTE: The duration of in vivo cytotoxicity can vary depending on the experimental system employed, the immunogenicity of target Ags, the anticipated abundance of peptide antigen-specific TCD8 in the spleen, and the robustness of their lytic function among other factors.</li>
	<li>Remove and process each spleen separately as in steps 3.2&#8722;3.9.</li>
	<li>Discard the supernatant and resuspend the pelleted cells in 3 mL of PBS. 
	NOTE: Take extra care to handle the splenic tissue and cell preparations at 4 &#176;C or on ice before cytofluorimetric analyses. This is to prevent continued cytotoxicity ex vivo.</li>
	<li>Transfer approximately 1 x 107 cells from each processed spleen into a clean FACS tube.</li>
	<li>Interrogate cells immediately using a flow cytometer equipped with a 488-nm laser. Draw a lymphocyte gate based on FSC and SSC properties of the cells.</li>
	<li>Identify CFSE- recipient&#8217;s splenocytes and CFSE+ transferred target cells. Draw additional gates accommodating distinct CFSElow, CFSEint, and CFSEhigh target cell populations.</li>
	<li>Acquire a total of 2000 CFSElow events in the FL-1 channel.</li>
</ol>
9. Data Analysis
<ol>
	<li>Calculate the specific lysis of each cognate target cell population using the following formula: 
	% Specific cytotoxicity = <img alt="Equation" src="/files/ftp_upload/59531/59531eq1.jpg" /> 
	where x = CFSEint/high event number in T Ag-primed mouse, y = CFSElow event number in T Ag-primed mouse, a = CFSEint/high event number in na&#239;ve mouse, and b = CFSElow event number in naive mouse. 
	NOTE: In &#8216;three-peak&#8217; cytotoxicity assays in which the specific lysis of more than one cognate target population is evaluated, it is not appropriate to use target cell frequencies. This is simply because the frequency of a cognate target cell population is influenced not only by the percentage of the irrelevant controls but also by that of the other cognate target splenocytes. Therefore, event numbers within each gate should be used in the above formula to accurately calculate the lysis of each cognate target cell population (either CFSEint or CFSEhigh cells) against CFSElow controls.</li>
</ol>

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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantification+of+Alloantibody-Mediated+Cytotoxicity+In+Vivo.">Quantification of Alloantibody-Mediated Cytotoxicity In Vivo.</a> Transplantation. 100 (5), 1041-1051 (2016).</li><li>Aichele, P., 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=Peptide+antigen+treatment+of+naive+and+virus-immune+mice:+antigen-specific+tolerance+versus+immunopathology.">Peptide antigen treatment of naive and virus-immune mice: antigen-specific tolerance versus immunopathology.</a> Immunity. 6 (5), 519-529 (1997).</li><li>Oehen, S., Brduscha-Riem, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differentiation+of+naive+CTL+to+effector+and+memory+CTL:+correlation+of+effector+function+with+phenotype+and+cell+division.">Differentiation of naive CTL to effector and memory CTL: correlation of effector function with phenotype and cell division.</a> The Journal of Immunology. 161 (10), 5338-5346 (1998).</li><li>Coles, R. M., Mueller, S. N., Heath, W. R., Carbone, F. R., Brooks, A. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Progression+of+armed+CTL+from+draining+lymph+node+to+spleen+shortly+after+localized+infection+with+herpes+simplex+virus+1.">Progression of armed CTL from draining lymph node to spleen shortly after localized infection with herpes simplex virus 1.</a> The Journal of Immunology. 168 (2), 834-838 (2002).</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> The Journal of Immunology. 171 (1), 27-31 (2003).</li><li>Meilleur, C. E., 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=Bacterial+superantigens+expand+and+activate,+rather+than+delete+or+incapacitate,+preexisting+antigen-specific+memory+CD8++T+cells.">Bacterial superantigens expand and activate, rather than delete or incapacitate, preexisting antigen-specific memory CD8+ T cells.</a> The Journal of Infectious Diseases. , (2018).</li><li>Goldszmid, R. 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=Dendritic+cells+charged+with+apoptotic+tumor+cells+induce+long-lived+protective+CD4++and+CD8++T+cell+immunity+against+B16+melanoma.">Dendritic cells charged with apoptotic tumor cells induce long-lived protective CD4+ and CD8+ T cell immunity against B16 melanoma.</a> The Journal of Immunology. 171 (11), 5940-5947 (2003).</li><li>Oberg, 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=Loss+or+mismatch+of+MHC+class+I+is+sufficient+to+trigger+NK+cell-mediated+rejection+of+resting+lymphocytes+in+vivo+-+role+of+KARAP/DAP12-dependent+and+-independent+pathways.">Loss or mismatch of MHC class I is sufficient to trigger NK cell-mediated rejection of resting lymphocytes in vivo - role of KARAP/DAP12-dependent and -independent pathways.</a> European Journal of Immunology. 34 (6), 1646-1653 (2004).</li><li>Wingender, G., Krebs, P., Beutler, B., Kronenberg, M. <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+cytotoxicity+by+invariant+NKT+cells+in+vivo+is+CD95/CD178-dependent+and+is+correlated+with+antigenic+potency.">Antigen-specific cytotoxicity by invariant NKT cells in vivo is CD95/CD178-dependent and is correlated with antigenic potency.</a> The Journal of Immunology. 185 (5), 2721-2729 (2010).</li><li>Brinster, R. 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=Transgenic+mice+harboring+SV40+T-antigen+genes+develop+characteristic+brain+tumors.">Transgenic mice harboring SV40 T-antigen genes develop characteristic brain tumors.</a> Cell. 37 (2), 367-379 (1984).</li><li>Tatum, A. M., 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=CD8++T+cells+targeting+a+single+immunodominant+epitope+are+sufficient+for+elimination+of+established+SV40+T+antigen-induced+brain+tumors.">CD8+ T cells targeting a single immunodominant epitope are sufficient for elimination of established SV40 T antigen-induced brain tumors.</a> The Journal of Immunology. 181 (6), 4406-4417 (2008).</li><li>Schell, T. D., Tevethia, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Control+of+advanced+choroid+plexus+tumors+in+SV40+T+antigen+transgenic+mice+following+priming+of+donor+CD8(+)+T+lymphocytes+by+the+endogenous+tumor+antigen.">Control of advanced choroid plexus tumors in SV40 T antigen transgenic mice following priming of donor CD8(+) T lymphocytes by the endogenous tumor antigen.</a> The Journal of Immunology. 167 (12), 6947-6956 (2001).</li><li>Greenberg, N. M., 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=Prostate+cancer+in+a+transgenic+mouse.">Prostate cancer in a transgenic mouse.</a> Proceedings of the National Academy of Sciences of the United States of America. 92 (8), 3439-3443 (1995).</li></ol>

The authors have nothing to disclose.

CFSE-based in vivo cytotoxicity assays offer several advantages over traditional killing assays such as radioactive chromium (51Cr) release and colorimetric lactate dehydrogenase (LDH) release assays. First, they permit the monitoring of CTL function within an architecturally intact secondary lymphoid organ.
Second, the specific killing of target cells in in vivo cytotoxicity assays reflects the absolute number of Ag-specific TCD8, which is usually, but not always, a function of TCD8 frequencies present in the spleen. This is in contrast with 51Cr/LDH release assays in which a constant number of cells are employed as a source of effector TCD8. Consequently, 51Cr/LDH release assays fail to reliably estimate the total number of Ag-specific TCD8 that may be available to the host to eliminate tumor cells or to combat infections. This is important since in many cases and conditions, the size and the cellularity of secondary lymphoid organs/tissues that accommodate Ag-specific TCD8 are altered. For instance, a hypothetical scenario can be envisaged in which a viral infection elevates the total number of TCD8 specific for peptide X while also expanding multiple other TCD8 clones harboring other specificities. As a result, the frequency of X-specific TCD8 among total splenic TCD8 may not increase, in which case a 51Cr/LDH release assay will not be helpful. As another example, we recently demonstrated that certain bacterial superantigens expand memory TCD8 specific for NP147-155, an immunodominant peptide epitope of influenza A viruses in BALB/c mice, which correlated well with increased in vivo lysis of NP147-155-pulsed target cells31. Since exposure to superantigens provokes T cell proliferation non-specifically, it would have been highly unlikely to demonstrate substantial NP147-155-specific cytotoxicity using 51Cr/LDH release assays.
Third, target cells pulsed with peptides that bind to the same MHC class I molecule can be labeled with different doses of CFSE, mixed and used in in vivo cytotoxicity assays. The concomitant analysis of CTL functions towards such peptides is not an option in 51Cr/LDH release assays.
Fourth, in vivo cytotoxicity assays allow for mechanistic studies during priming, effector and recall phases of CTL responses. For example, tumor cell inoculation or antitumor vaccination can be conducted in genetically altered mice for the assessment of CTL induction. Moreover, splenocytes from gene knock-in and knock-out mice can be used as target cells during the effector phase. Finally, various agents (e.g., pharmacological inhibitors and drug candidates) can be administered before priming, during the effector phase, or both. Therefore, in vivo cytotoxicity assays provide a powerful platform for drug/vaccine efficacy testing in a truly in vivo setting. In this body of work, we have provided examples of immunological interventions that boost in vivo CTL responses (Figure 1 and Figure 2).
Like other routinely used killing assays, in vivo cytotoxicity assays do not provide any direct information regarding the ability of CTLs to recycle from one target to another before they become exhausted. In addition, we have tested several tumor cell types as potential target cells in in vivo cytotoxicity assays, albeit to no avail so far. This is simply because tumor cells do not reach the spleen at least in detectable numbers after they are injected i.v. Therefore, relying on mouse splenocytes as target cells may be considered an inherent limitation of in vivo cytotoxicity assays. It is noteworthy, however, that adoptively transferred target splenocytes can be easily found in several other organs (in addition to the spleen), for instance in the liver. Therefore, Ag-specific CTL function can be assessed in multiple organs or tissues.
We have optimized in vivo cytotoxicity assays for the examination of immunodominance in T Ag-specific TCD8 responses16,17. Numerous tools and reagents are available for studying these responses in the contexts of antitumor immunity and therapy. The fibrosarcoma cell line used in the protocol described here (i.e., C57SV cells) does not give rise to tumors in immunocompetent mice. Therefore, it is a useful tool in investigating antitumor vaccination. T Ag-driven neoplastic transformation in select tissues has generated several valuable models of autochthonous cancer. For example, SV11 mice that develop choroid plexus papillomas inside their brain ventricles35 do not harbor endogenous T Ag-specific TCD8 because these cells are selected against and deleted in the thymus. However, transferring C57BL/6 splenocytes into sublethally irradiated, tumor-bearing SV11 mice leads to extended control of the tumors, which is reportedly associated with in vivo priming of site IV-specific TCD836,37. In the transgenic adenocarcinoma of the mouse prostate (TRAMP) model38, the site IV-specific response dwindles away with progression of the malignancy. However, the otherwise immunorecessive site V-specific TCD8 cells escape negative selection in the thymus and also avoid peripheral tolerance mechanisms21. This provides ample opportunities for experimental therapeutic interventions revolving around site V-specific TCD8 functions. In vivo cytotoxicity assays should prove informative in studying and potentially reversing immunological tolerance in various model systems, including in the T Ag recognition model.
Immunodominance is a consistent feature of TCD8 responses generated not only against tumor Ags but also towards pathogen-derived epitopes. In fact, we have previously used in vivo cytotoxicity assays to study immunodominance in anti-influenza TCD8 responses12. Therefore, the optimized assay described in this protocol can be modified and used in a broad range of immunological applications.

Conventional CD8+ T cells (TCD8) play important parts in anticancer immune surveillance. They primarily function in the capacity of cytolytic T lymphocytes (CTLs) that recognize tumor-specific or -associated peptide antigens (Ags) displayed within the closed cleft of major histocompatibility complex (MHC) class I molecules. Fully armed CTLs utilize their cytotoxic arsenal to destroy malignant cells. Anticancer TCD8 can be detected in the circulation or even inside primary and metastatic masses of many cancer patients and tumor-bearing animals. However, they are often anergic or exhausted and fail to eradicate cancer. Therefore, many immunotherapeutic modalities are designed to increase anticancer TCD8 frequencies and to restore and boost their functions.
Tumor proteins harbor many peptides, some of which can be immunogenic and potentially immunoprotective. However, quantifiable TCD8 responses are elicited with varying magnitudes against few peptides only. This creates an &#8220;immunodominance hierarchy&#8221; among TCD8 clones1. Accordingly, immunodominant (ID) TCD8 occupy prominent hierarchical ranks, which is commonly judged by their abundance. In contrast, TCD8 cells whose T cell receptor (TCR) is specific for subdominant (SD) epitopes occur in lower frequencies. We and others have identified some of the factors that dictate or shape immunodominance in TCD8 responses. These include, among others, the mode of Ag presentation to na&#239;ve TCD8 (i.e., direct presentation, cross-presentation, cross-dressing)2,3,4, the type of Ag-presenting cells (APCs) participating in TCD8 activation5, the abundance and stability of protein Ags6,7 and the efficiency and kinetics of their degradation by proteasomes7,8, the relative selectivity of transporter associated with Ag processing (TAP) for peptides9, the affinity of liberated peptides for MHC I molecules9,10, the presence, precursor frequencies and TCR diversity of cognate TCD8 in T cell pools11,12,13, cross-competition among T cells for access to APCs14,15, and the fratricidal capacity of TCD8 clones16. In addition, TCD8 immunodominance is subjected to immunoregulatory mechanisms mediated by several suppressor cell types such as naturally occurring regulatory T (nTreg) cells17, the cell surface co-inhibitory molecule programmed death-1 (PD-1)16, and certain intracellular enzymes such as indoleamine 2,3-dioxygenase (IDO)18 and the mammalian target of rapamycin (mTOR)19. It is important to note, however, that the above factors do not always fully account for immunodominance.
Apart from the basic biology of TCD8 immunodominance, the examination of this intriguing phenomenon has important implications in cancer immunology and immunotherapy. First, an ID status does not necessarily confer upon a given TCD8 clone the ability to prevent tumor initiation or progression20. Whether and how ID and SD TCD8 contribute to antitumor immunity may be dependent upon the type and the extent of malignancy and the experimental system employed. Second, it is thought that ID TCD8 clones may be &#8216;too visible&#8217; to the immune system and consequently more prone to central and/or peripheral tolerance mechanisms16,21. Third, heterogeneic tumors may contain neoplastic cells that avoid detection by many, if not most, CTLs by displaying only a narrow spectrum of peptide:MHC complexes. Under these circumstances, TCD8 responses of insufficient breadth are likely to afford such tumor cells a survival advantage, thus potentiating their outgrowth22. It is for the above reasons that many view immunodominance as a hurdle to successful TCD8-based vaccination and therapies against cancer.
Inoculation of C57BL/6 mice with simian virus 40 (SV40)-transformed cells that express large tumor Ag (T Ag) provides a powerful preclinical system to study TCD8 immunodominance. This model offers several benefits. First, the peptide epitopes of this clinically relevant oncoprotein are well-characterized in this mouse strain23 (Table 1). Second, T Ag epitopes, which are called sites I, II/III, IV, and V, trigger TCD8 responses that are consistently arranged in the following hierarchical order: site IV &#62;&#62; site I &#8805; site II/III &#62;&#62; site V. Accordingly, site IV-specific TCD8 mount the most robust response to T Ag. In contrast, sites I and II/III are subdominant, and site V-specific TCD8 are least abundant and usually only detectable in the absence of responsiveness to other epitopes23,24. Third, the T Ag+ tumor cell line utilized in the protocol described herein, namely C57SV fibrosarcoma cells, and those used in our previous investigations16,17,18,19,25,26, are transformed with subgenomic SV40 fragments25. Therefore, they are unable to assemble and release SV40 virions that could potentially infect host APCs. In addition, C57SV cells are devoid of classic costimulatory molecules such as CD80 (B7-1), CD86 (B7-2), and CD137 ligand (4-1BBL)16. The above attributes make these lines ideal for examination of in vivo TCD8 activation via cross-priming. Cross-priming is a major pathway in inducing TCD8 responses, especially those launched against tumor cells of non-hematopoietic origin that fail to directly prime na&#239;ve T cells25.
Antitumor TCD8 frequencies and/or functions can be monitored by MHC I tetramer staining, intracellular staining for effector cytokines (e.g., interferon [IFN]-&#947;) or lytic molecules (e.g., perforin), enzyme-linked immunospot (ELISpot) assays and ex vivo cytotoxicity assays. Since their inception in the 1990s27,28, carboxyfluorescein succinimidyl ester (CFSE)-based in vivo killing assays have enabled evaluation of cytotoxic responses mediated by antiviral CTLs29,30,31, antitumor CTLs16,32, natural killer (NK) cells33, glycolipid-reactive invariant natural killer T (iNKT) cells34, and preexisting and de novo donor-specific alloantibodies26. Therefore, their applications can be of interest to a wide readership, including but not limited to investigators working in the areas of tumor immunology and immunotherapy, anti-pathogen immunity, and preventative and therapeutic vaccine design.&#160;&#160;
To assess cell-mediated cytotoxicity in typical scenarios, two populations of na&#239;ve splenocytes that display either an irrelevant Ag or a cognate Ag(s) are labeled with two different doses of CFSE, mixed in equal numbers and injected into na&#239;ve (control) or killer cell-harboring mice. The presence/absence of each target population is then examined by flow cytometry.&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;
We have optimized and employed in vivo killing assays in our studies on immunodominance in both antiviral and antitumor TCD8 responses12,16,17. Here, we provide a detailed protocol for the simultaneous assessment of ID and SD TCD8 responses to T Ag epitopes, which can be readily adopted for similar investigations in other experimental systems. We also provide representative results demonstrating that nTreg cell depletion and PD-1 blockade can selectively enhance ID TCD8- and SD TCD8-induced cytotoxicity, respectively. At the end, we will discuss multiple advantages of in vivo killing assays as well as some of their inherent limitations.

<table border="0" cellpadding="0" cellspacing="0" style="width:872px;" width="871">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">0.25% Trypsin-EDTA (1X)</td>
			<td style="width:164px;">Thermo Fisher Scientific</td>
			<td style="width:163px;">25200-056</td>
			<td style="width:211px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ACK Lysing Buffer</td>
			<td>Thermo Fisher Scientific</td>
			<td style="width:163px;">A1049201</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-mouse CD25 (clone PC-61.5.3)</td>
			<td>Bio X Cell</td>
			<td>BE0012</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-mouse PD-1 (clone RMP1-14)</td>
			<td>Bio X Cell</td>
			<td>BE0146</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CFSE</td>
			<td>Thermo Fisher Scientific</td>
			<td>C34554</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DMEM (1X)</td>
			<td>Thermo Fisher Scientific</td>
			<td>11965-092</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fetal bovine serum (FBS)</td>
			<td>Wisent Bioproducts</td>
			<td>080-150</td>
			<td>Heat-inactivate prior to use</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">GlutaMAX (100X)</td>
			<td>Thermo Fisher Scientific</td>
			<td>35050-061</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">HEPES (1M)</td>
			<td>Thermo Fisher Scientific</td>
			<td>15630080</td>
			<td>10 mM final concentration</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MEM Non-Essential Amino Acids Solution (100X)&#160;</td>
			<td>Thermo Fisher Scientific</td>
			<td>11140-050</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Penicillin/Streptomycin</td>
			<td>Sigma-Aldrich</td>
			<td>P0781</td>
			<td>Stock is 100X</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Rat IgG1 (clone KLH/G1-2-2)</td>
			<td>SouthernBiotech</td>
			<td>0116-01</td>
			<td>Isotype control</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Rat IgG1 (clone HRPN)</td>
			<td>Bio X Cell</td>
			<td>BE0088</td>
			<td>Isotype control</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Rat IgG1 (clone TNP6A7)</td>
			<td>Bio X Cell</td>
			<td>BP0290</td>
			<td>Isotype control</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Rat IgG2a (clone 2A3)</td>
			<td>Bio X Cell</td>
			<td>BP0089</td>
			<td>Isotype control</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">RPMI 1640 (1X)</td>
			<td>Thermo Fisher Scientific</td>
			<td>11875-093</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium Pyruvate (100 mM)</td>
			<td>Thermo Fisher Scientific</td>
			<td>11360-070</td>
			<td>1 mM final concentration</td>
		</tr>
	</tbody>
</table>

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.

The goal of the experiment whose results are depicted in Figure 1 was to determine whether the presence and functions of nTreg cells shape or alter the immunodominance hierarchy of T Ag-specific TCD8. C57BL/6 mice were injected i.p. with PBS or with 0.5 mg of an anti-CD25 mAb (clone PC-61.5.3 [PC61]) four days before they received 2 x 107 C57SV tumor cells i.p. In separate experiments, a rat IgG1 isotype control was used in lieu of PBS. Successful nTreg cell depletion by PC61 was confirmed by flow cytometry17.
Nine days after C57SV cell inoculation, a time point at which T Ag-specific TCD8 responses reach their maximum23, each animal received an i.v. injection of a cell suspension containing 3 distinct populations of CFSE-labeled target cells. Control target cells were syngeneic na&#239;ve splenocytes concomitantly pulsed with two irrelevant peptides (GP33-41 and gB498-505) and labeled with a low dose of CFSE (0.02 &#956;M). To prepare cognate target cells, syngeneic na&#239;ve splenocytes were pulsed with either T Ag-derived site I peptide or site IV peptide (Table 1), and subsequently labeled with CFSE at 0.2 &#956;M and 2 &#956;M, respectively. Control and cognate target cells were washed and mixed in equal numbers (at a 1:1:1 ratio) before they were injected into na&#239;ve (control) and T Ag-primed C57BL/6 mice. Two hours after target cell injection, mice were sacrificed for their spleen in which the presence/absence of CFSE-labeled target cells was determined by flow cytometry. Target cells were distinguished based on their differential CFSE staining intensities.
As expected, near-equal peaks corresponding to control and cognate target cells were detectable in na&#239;ve mice (Figure 1, left panel). In contrast, site IV-displaying target cells were almost completely absent in T Ag-primed mice regardless of their prior treatment with PC61 or PBS (Figure 1). Interestingly, nTreg cell depletion by PC61 augmented in vivo CTL-mediated lysis of site I-pulsed target cells17. These results prompted us to conclude that nTreg cells selectively inhibit site I-specific cytotoxicity. Therefore, nTreg cell-depleting/inactivating agents may enhance the cytolytic effector function of CTLs recognizing certain tumor-derived epitopes.
The above set-up provides an example of how in vivo cytotoxicity assays can be employed to simultaneously test the lytic function of ID and SD CTL clones in the same animal.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/59531/59531fig1.jpg" /> 
Figure 1: Representative cytofluorimetric analysis of TCD8-mediated cytotoxicity against T Ag-derived epitopes in the presence or absence of nTreg cells. Target splenocytes pulsed with control peptides, site I or site IV, which were differentially labeled with CFSE, were tracked by flow cytometry in the spleen of a na&#239;ve mouse (left panel), a PBS-injected T Ag-primed mouse (middle panel), and a PC61 (nti-CD25)-injected (nTreg-depleted) T Ag-primed mouse (right panel). Percent specific killing of target cells was calculated using the formula described in the protocol, and representative numbers are shown. This figure is adopted, with permission, from Haeryfar et al.17. Copyright 2005. The American Association of Immunologists, Inc. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59531/59531fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
In a more recent investigation, we asked whether blocking PD-1 affects the &#8216;breadth&#8217; of the TCD8 response to T Ag16 (Figure 2). This was a clinically relevant question in light of the observed therapeutic benefits of PD-1-based &#8216;checkpoint inhibitors&#8217; in several malignancies. Although such inhibitors are thought to work primarily by reversing T cell exhaustion, we were curious to know whether interfering with PD-1-PD-L1 interactions may additionally widen (or narrow) anticancer TCD8 responses. In our T Ag recognition model, intracellular cytokine staining (ICS) experiments revealed that treatment with either anti-PD-1 or anti-PD-L-1 selectively expands IFN-&#947;-producing TCD8 recognizing sites I and II/III16. We then extended our study to examine the in vivo cytolytic effector function of these SD CTLs. C57BL/6 mice were injected i.p. with 100 &#956;g of an anti-PD-1 mAb (clone RMP1-14) or a rat IgG2a isotype control (clone 2A3) two hours before C57SV cell inoculation. Mice received two additional doses of anti-PD-1 or isotype three and six days after tumor cell injection. On day 9 post-priming, cohorts of na&#239;ve and primed mice were given, via lateral tail veins, a cell mixture containing equal numbers of CFSElow (CFSE labeling dose: 0.025 &#956;M), CFSEint (CFSE labeling dose: 0.25 &#956;M), and CFSEhi (CFSE labeling dose: 2 &#956;M) syngeneic na&#239;ve splenocytes pulsed with gB498-505, site II/III, and site I, respectively. Four hours later, animals were euthanized, and CFSE-labeled target cells were tracked cytofluorimetrically in their spleen. Representative FACS plots (Figure 2A) and data from 3 animals per cohort (Figure 2B) are illustrated. While PD-1 blockade did not affect the ID TCD8 response against site IV16, sites I- and II/III-specific SD responses were invigorated. We thus concluded that interfering with PD-1-PD-L1 interactions may induce &#8216;epitope spreading&#8217; in anticancer TCD8 responses.
The above set-up represents in vivo killing assays that enable quantitation of cytotoxicity elicited by two SD CTL clones in the same animal.
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/59531/59531fig2.jpg" /> 
Figure 2: In vivo cytotoxicity of T Ag-specific TCD8 in anti-PD-1-treated mice. (A) Representative histogram plots demonstrate CFSE peaks corresponding to target splenocytes pulsed with an irrelevant peptide (CFSElow), site II/III (CFSEint), and site I (CFSEhigh) in T Ag-primed mice that received an isotype (left panel) or a PD-1-blocking mAb (right panel). (B) Percent specific killing of each cognate target cell population was calculated using CFSE+ event numbers in T Ag-primed mice (n = 3 per group) and na&#239;ve recipients (not shown) and the formula described in the protocol. Error bars represent standard errors of the mean (SEM), and ** denotes a statistical difference with p &#60; 0.01 by unpaired Student&#8217;s t-tests. This figure is adopted, with permission, from Memarnejadian et al.16. Copyright 2017. The American Association of Immunologists, Inc. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59531/59531fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Protein Antigen Source</td>
			<td>Peptide Epitope</td>
			<td>Designation</td>
			<td>Sequence</td>
			<td>MHC I Restriction</td>
		</tr>
		<tr>
			<td>SV401 Large T Ag2</td>
			<td>T Ag206-215</td>
			<td>Site I</td>
			<td>SAINNYAQKL</td>
			<td>H-2Db</td>
		</tr>
		<tr>
			<td>SV40 Large T Ag</td>
			<td>T Ag223-231</td>
			<td>Site II/III</td>
			<td>CKGVNKEYL</td>
			<td>H-2Db</td>
		</tr>
		<tr>
			<td>SV40 Large T Ag</td>
			<td>T Ag404-411</td>
			<td>Site IV</td>
			<td>VVYDFLKC</td>
			<td>H-2Kb</td>
		</tr>
		<tr>
			<td>SV40 Large T Ag</td>
			<td>T Ag489-497</td>
			<td>Site V</td>
			<td>QGINNLDNL</td>
			<td>H-2Db</td>
		</tr>
		<tr>
			<td>HSV-13 Glycoprotein B</td>
			<td>gB498-505*</td>
			<td>gB498-505</td>
			<td>SSIEFARL</td>
			<td>H-2Kb</td>
		</tr>
		<tr>
			<td>LCMV4 Glycoprotein</td>
			<td>GP33-41*</td>
			<td>GP33-41</td>
			<td>KAVYNFATC</td>
			<td>H-2Db</td>
		</tr>
		<tr>
			<td>1Simian Virus 40</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>2Large Tumor Antigen</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td colspan="2">3Herpes Simplex Virus type 1</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td colspan="2">4Lymphocytic Choriomeningitis Virus</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td colspan="2">*used as an irrelevant peptide</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>
Table 1. Peptides introduced in this protocol

Watch this Scientific Journal Video about Tailoring In Vivo Cytotoxicity Assays to Study Immunodominance in Tumor-specific CD8+ T Cell Responses at JoVE.com

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.

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 ...

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Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

8.9K Views.  Western University. 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.

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

Retroviral Transduction of T-cell Receptors in Mouse T-cells

T-cell receptors (TCRs) play a central role in the immune system. TCRs on T-cell surfaces can specifically recognize peptide antigens presented by antigen presenting cells (APCs)1. This recognition leads to the activation of T-cells and a series of functional outcomes (e.g. cytokine production, killing of the target cells). Understanding the functional role of TCRs is critical to harness the power of the immune system to treat a variety of immunology related diseases (e.g. cancer or autoimmunity).
It is convenient to study TCRs in mouse models, which can be accomplished in several ways. Making TCR transgenic mouse models is costly and time-consuming and currently there are only a limited number of them available2-4. Alternatively, mice with antigen-specific T-cells can be generated by bone marrow chimera. This method also takes several weeks and requires expertise5. Retroviral transduction of TCRs into in vitro activated mouse T-cells is a quick and relatively easy method to obtain T-cells of desired peptide-MHC specificity. Antigen-specific T-cells can be generated in one week and used in any downstream applications. Studying transduced T-cells also has direct application to human immunotherapy, as adoptive transfer of human T-cells transduced with antigen-specific TCRs is an emerging strategy for cancer treatment6.
Here we present a protocol to retrovirally transduce TCRs into in vitro activated mouse T-cells. Both human and mouse TCR genes can be used. Retroviruses carrying specific TCR genes are generated and used to infect mouse T-cells activated with anti-CD3 and anti-CD28 antibodies. After in vitro expansion, transduced T-cells are analyzed by flow cytometry.

We present a protocol to produce antigen-specific mouse T-cells using retroviral transduction

T-cell receptors (TCRs) play a central role in the immune system. TCRs on T-cell surfaces can specifically recognize peptide antigens presented by antigen ...

Competitive Homing Assays to Study Gut-tropic T Cell Migration

In order to exert their function lymphocytes need to leave the blood and migrate into different tissues in the body. Lymphocyte adhesion to endothelial cells and tissue extravasation is a multistep process controlled by different adhesion molecules (homing receptors) expressed on lymphocytes and their respective ligands (addressins) displayed on endothelial cells 1 2. Even though the function of these adhesion receptors can be partially studied ex vivo, the ultimate test for their physiological relevance is to assess their role during in vivo lymphocyte adhesion and migration. Two complementary strategies have been used for this purpose: intravital microscopy (IVM) and homing experiments. Although IVM has been essential to define the precise contribution of specific adhesion receptors during the adhesion cascade in real time and in different tissues, IVM is time consuming and labor intensive, it often requires the development of sophisticated surgical techniques, it needs prior isolation of homogeneous cell populations and it permits the analysis of only one tissue/organ at any given time. By contrast, competitive homing experiments allow the direct and simultaneous comparison in the migration of two (or even more) cell subsets in the same mouse and they also permit the analysis of many tissues and of a high number of cells in the same experiment.
Here we describe the classical competitive homing protocol used to determine the advantage/disadvantage of a given cell type to home to specific tissues as compared to a control cell population. We chose to illustrate the migratory properties of gut-tropic versus non gut-tropic T cells, because the intestinal mucosa is the largest body surface in contact with the external environment and it is also the extra-lymphoid tissue with the best-defined migratory requirements. Moreover, recent work has determined that the vitamin A metabolite all-trans retinoic acid (RA) is the main molecular mechanism responsible for inducing gut-specific adhesion receptors (integrin a4b7and chemokine receptor CCR9) on lymphocytes. Thus, we can readily generate large numbers of gut-tropic and non gut-tropic lymphocytes ex vivoby activating T cells in the presence or absence of RA, respectively, which can be finally used in the competitive homing experiments described here.

Competitive homing experiments allow to directly assessing the migratory properties of two different cell populations in a single mouse. Here we illustrate this procedure by comparing the migration of ex vivo-generated gut-tropic versus non-gut tropic T cells.

In order to exert their function lymphocytes need to leave the blood and migrate into different tissues in the body. Lymphocyte adhesion to endothelial ...

piggyBac Transposon System Modification of Primary Human T Cells

The piggyBac transposon system is naturally active, originally derived from the cabbage looper moth1,2. This non-viral system is plasmid based, most commonly utilizing two plasmids with one expressing the piggyBac transposase enzyme and a transposon plasmid harboring the gene(s) of interest between inverted repeat elements which are required for gene transfer activity. PiggyBac mediates gene transfer through a "cut and paste" mechanism whereby the transposase integrates the transposon segment into the genome of the target cell(s) of interest. PiggyBac has demonstrated efficient gene delivery activity in a wide variety of insect1,2, mammalian3-5, and human cells6 including primary human T cells7,8. Recently, a hyperactive piggyBac transposase was generated improving gene transfer efficiency9,10.
Human T lymphocytes are of clinical interest for adoptive immunotherapy of cancer11. Of note, the first clinical trial involving transposon modification of human T cells using the Sleeping beauty transposon system has been approved12. We have previously evaluated the utility of piggyBac as a non-viral methodology for genetic modification of human T cells. We found piggyBac to be efficient in genetic modification of human T cells with a reporter gene and a non-immunogenic inducible suicide gene7. Analysis of genomic integration sites revealed a lack of preference for integration into or near known proto-oncogenes13. We used piggyBac to gene-modify cytotoxic T lymphocytes to carry a chimeric antigen receptor directed against the tumor antigen HER2, and found that gene-modified T cells mediated targeted killing of HER2-positive tumor cells in vitro and in vivo in an orthotopic mouse model14. We have also used piggyBac to generate human T cells resistant to rapamycin, which should be useful in cancer therapies where rapamycin is utilized15.
Herein, we describe a method for using piggyBac to genetically modify primary human T cells. This includes isolation of peripheral blood mononuclear cells (PBMCs) from human blood followed by culture, gene modification, and activation of T cells. For the purpose of this report, T cells were modified with a reporter gene (eGFP) for analysis and quantification of gene expression by flow cytometry.
PiggyBac can be used to modify human T cells with a variety of genes of interest. Although we have used piggyBac to direct T cells to tumor antigens14, we have also used piggyBac to add an inducible safety switch in order to eliminate gene modified cells if needed7. The large cargo capacity of piggyBac has also enabled gene transfer of a large rapamycin resistant mTOR molecule (15 kb)15. Therefore, we present a non-viral methodology for stable gene-modification of primary human T cells for a wide variety of purposes.

piggyBac Transposon System Modification of Primary Human T Cells

We describe a method to genetically modify primary human T cells with a transgene using the non-viral piggyBac transposon system. T cells modified to using the piggyBac transposon system exhibit stable transgene expression.

The piggyBac transposon system is naturally active, originally derived from the cabbage looper moth1,2. This non-viral system is plasmid based, most ...

Quantitative High-throughput Single-cell Cytotoxicity Assay For T Cells

Cancer immunotherapy can harness the specificity of immune response to target and eliminate tumors. Adoptive cell therapy (ACT) based on the adoptive transfer of T cells genetically modified to express a chimeric antigen receptor (CAR) has shown considerable promise in clinical trials1-4. There are several advantages to using CAR+ T cells for the treatment of cancers including the ability to target non-MHC restricted antigens and to functionalize the T cells for optimal survival, homing and persistence within the host; and finally to induce apoptosis of CAR+ T cells in the event of host toxicity5.
Delineating the optimal functions of CAR+ T cells associated with clinical benefit is essential for designing the next generation of clinical trials. Recent advances in live animal imaging like multiphoton microscopy have revolutionized the study of immune cell function in vivo6,7. While these studies have advanced our understanding of T-cell functions in vivo, T-cell based ACT in clinical trials requires the need to link molecular and functional features of T-cell preparations pre-infusion with clinical efficacy post-infusion, by utilizing in vitro assays monitoring T-cell functions like, cytotoxicity and cytokine secretion. Standard flow-cytometry based assays have been developed that determine the overall functioning of populations of T cells at the single-cell level but these are not suitable for monitoring conjugate formation and lifetimes or the ability of the same cell to kill multiple targets8.
Microfabricated arrays designed in biocompatible polymers like polydimethylsiloxane (PDMS) are a particularly attractive method to spatially confine effectors and targets in small volumes9. In combination with automated time-lapse fluorescence microscopy, thousands of effector-target interactions can be monitored simultaneously by imaging individual wells of a nanowell array. We present here a high-throughput methodology for monitoring T-cell mediated cytotoxicity at the single-cell level that can be broadly applied to studying the cytolytic functionality of T cells.

We describe a single-cell high-throughput assay to measure cytotoxicity of T cells when incubated with tumor target cells. This method employs a dense, elastomeric array of sub-nanoliter wells (~100,000 wells/array) to spatially confine the T cells and target cells at defined ratios and is coupled to fluorescence microscopy to monitor effector-target conjugation and subsequent apoptosis.

Cancer immunotherapy can harness the specificity of  immune response to target and eliminate tumors. Adoptive cell therapy (ACT)  based on the adoptive ...

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 ...

Transplantation of Tail Skin to Study Allogeneic CD4 T Cell Responses in Mice

The study of T cell responses and their consequences during allo-antigen recognition requires a model that enables one to distinguish between donor and host T cells, to easily monitor the graft, and to adapt the system in order to answer different immunological questions. Medawar and colleagues established allogeneic tail-skin transplantation in mice in 1955. Since then, the skin transplantation model has been continuously modified and adapted to answer specific questions. The use of tail-skin renders this model easy to score for graft rejection, requires neither extensive preparation nor deep anesthesia, is applicable to animals of all genetic background, discourages ischemic necrosis, and permits chemical and biological intervention. 
In general, both CD4+ and CD8+ allogeneic T cells are responsible for the rejection of allografts since they recognize mismatched major histocompatibility antigens from different mouse strains. Several models have been described for activating allogeneic T cells in skin-transplanted mice. The identification of major histocompatibility complex (MHC) class I and II molecules in different mouse strains including C57BL/6 mice was an important step toward understanding and studying T cell-mediated alloresponses. In the tail-skin transplantation model described here, a three-point mutation (I-Abm12) in the antigen-presenting groove of the MHC-class II (I-Ab) molecule is sufficient to induce strong allogeneic CD4+ T cell activation in C57BL/6 mice. Skin grafts from I-Abm12 mice on C57BL/6 mice are rejected within 12-15 days, while syngeneic grafts are accepted for up to 100 days. The absence of T cells (CD3-/- and Rag2-/- mice) allows skin graft acceptance up to 100 days, which can be overcome by transferring 2 x 104 wild type or transgenic T cells. Adoptively transferred T cells proliferate and produce IFN-&#947; in I-Abm12-transplanted Rag2-/- mice.

Tail-skin transplantation is a powerful model for studying T cell-dependent rejection and tolerance induction during allogeneic immune responses in mice. The advantages of this protocol are minor invasive surgery, and ease of monitoring with no need to sacrifice the recipient mouse.

The study of T cell responses and their consequences during allo-antigen recognition requires a model that enables one to distinguish between donor and ...

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 ...

Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

Salmonella typhimurium is a facultative intracellular bacterium that causes gastroenteritis in humans. After invasion of the lamina propria, S. typhimurium bacteria are quickly detected and phagocytized by macrophages, and contained in vesicles known as phagosomes in order to be degraded. Isolation of S. typhimurium-containing phagosomes have been widely used to study how S. typhimurium infection alters the process of phagosome maturation to prevent bacterial degradation. Classically, the isolation of bacteria-containing phagosomes has been performed by sucrose gradient centrifugation. However, this process is time-consuming, and requires specialized equipment and a certain degree of dexterity. Described here is a simple and quick method for the isolation of S. typhimurium-containing phagosomes from macrophages by coating the bacteria with biotin-streptavidin-conjugated magnetic beads. Phagosomes obtained by this method can be suspended in any buffer of choice, allowing the utilization of isolated phagosomes for a broad range of assays, such as protein, metabolite, and lipid analysis. In summary, this method for the isolation of S. typhimurium-containing phagosomes is specific, efficient, rapid, requires minimum equipment, and is more versatile than the classical method of isolation by sucrose gradient-ultracentrifugation.

Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

We describe here a simple and quick method for the isolation of Salmonella typhimurium-containing phagosomes from macrophages by coating the bacteria with biotin and streptavidin.

Salmonella typhimurium is a facultative intracellular bacterium that causes gastroenteritis in humans. After invasion of the lamina propria, S. ...

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 ...

Characterization of Thymus-dependent and Thymus-independent Immunoglobulin Isotype Responses in Mice Using Enzyme-linked Immunosorbent Assay

Antibodies, also termed as immunoglobulins (Ig), secreted by differentiated B lymphocytes, plasmablasts/plasma cells, in humoral immunity provide a formidable defense against invading pathogens via diverse mechanisms. One major goal of vaccination is to induce protective antigen-specific antibodies to prevent life-threatening infections. Both thymus-dependent (TD) and thymus-independent (TI) antigens can elicit robust antigen-specific IgM responses and can also induce the production of isotype-switched antibodies (IgG, IgA and IgE) as well as the generation of memory B cells with the help provided by antigen presenting cells (APCs). Here, we describe a protocol to characterize TD and TI Ig isotype responses in mice using enzyme-linked immunosorbent assay (ELISA). In this protocol, TD and TI Ig responses are elicited in mice by intraperitoneal (i.p.) immunization with hapten-conjugated model antigens TNP-KLH (in alum) and TNP-polysaccharide (in PBS), respectively. To induce TD memory response, a booster immunization of TNP-KLH in alum is given at 3 weeks after the first immunization with the same antigen/adjuvant. Mouse sera are harvested at different time points before and after immunization. Total serum Ig levels and TNP-specific antibodies are subsequently quantified using Ig isotype-specific Sandwich and indirect ELISA, respectively. In order to correctly quantify the serum concentration of each Ig isotype, the samples need to be appropriately diluted to fit within the linear range of the standard curves. Using this protocol, we have consistently obtained reliable results with high specificity and sensitivity. When used in combination with other complementary methods such as flow cytometry, in vitro culture of splenic B cells and immunohistochemical staining (IHC), this protocol will allow researchers to gain a comprehensive understanding of antibody responses in a given experimental setting.

In this paper, we describe a protocol to characterize T-dependent and T-independent immunoglobulin (Ig) isotype responses in mice using ELISA. This method used alone or in combination with flow cytometry will allow researchers to identify differences in B cell-mediated Ig isotype responses in mice following T-dependent and T-independent antigen immunization.

Antibodies, also termed as immunoglobulins (Ig), secreted by differentiated B lymphocytes, plasmablasts/plasma cells, in humoral immunity provide a ...

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

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Chapters in this video

Retroviral Transduction of T-cell Receptors in Mouse T-cells

Competitive Homing Assays to Study Gut-tropic T Cell Migration

piggyBac Transposon System Modification of Primary Human T Cells

Quantitative High-throughput Single-cell Cytotoxicity Assay For T Cells

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

Transplantation of Tail Skin to Study Allogeneic CD4 T Cell Responses in Mice

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

Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

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

Characterization of Thymus-dependent and Thymus-independent Immunoglobulin Isotype Responses in Mice Using Enzyme-linked Immunosorbent Assay