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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D., Haeryfar, S. 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 func...

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><tbody><tr><td>0.25% Trypsin-EDTA (1X)</td><td>Thermo Fisher Scientific</td><td>25200-056</td><td></td></tr><tr><td>ACK Lysing Buffer</td><td>Thermo Fisher Scientific</td><td>A1049201</td><td></td></tr><tr><td>Anti-mouse CD25 (clone PC-61.5.3)</td><td>Bio X Cell</td><td>BE0012</td><td></td></tr><tr><td>Anti-mouse PD-1 (clone RMP1-14)</td><td>Bio X Cell</td><td>BE0146</td><td></td></tr><tr><td>CFSE</td><td>Thermo Fisher Scientific</td><td>C34554</td><td></td></tr><tr><td>DMEM (1X)</td><td>Thermo Fisher Scientific</td><td>11965-092</td><td></td></tr><tr><td>Fetal bovine serum (FBS)</td><td>Wisent Bioproducts</td><td>080-150</td><td>Heat-inactivate prior to use</td></tr><tr><td>GlutaMAX (100X)</td><td>Thermo Fisher Scientific</td><td>35050-061</td><td></td></tr><tr><td>HEPES (1M)</td><td>Thermo Fisher Scientific</td><td>15630080</td><td>10 mM final concentration</td></tr><tr><td>MEM Non-Essential Amino Acids Solution (100X)&amp;nbsp;</td><td>Thermo Fisher Scientific</td><td>11140-050</td><td></td></tr><tr><td>Penicillin/Streptomycin</td><td>Sigma-Aldrich</td><td>P0781</td><td>Stock is 100X</td></tr><tr><td>Rat IgG1 (clone KLH/G1-2-2)</td><td>SouthernBiotech</td><td>0116-01</td><td>Isotype control</td></tr><tr><td>Rat IgG1 (clone HRPN)</td><td>Bio X Cell</td><td>BE0088</td><td>Isotype control</td></tr><tr><td>Rat IgG1 (clone TNP6A7)</td><td>Bio X Cell</td><td>BP0290</td><td>Isotype control</td></tr><tr><td>Rat IgG2a (clone 2A3)</td><td>Bio X Cell</td><td>BP0089</td><td>Isotype control</td></tr><tr><td>RPMI 1640 (1X)</td><td>Thermo Fisher Scientific</td><td>11875-093</td><td></td></tr><tr><td>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 b...

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 ...

tailoring-vivo-cytotoxicity-assays-to-study-immunodominance-tumor

Research

JoVE Journal

Immunology and Infection

8.9K Views.  Western University. Deze test kan worden gebruikt om T-cel-gemedieerde cytotoxiciteit kwantificeren tegen twee verschillende peptide epitopen van tumor antigenen tegelijkertijd. De in vivo aard van deze test maakt het mogelijk om cytotoxische functie van T-cellen te meten binnen de intacte architectuur van een secundair lymfeorgaan. Bovendien is het waargenomen doden een nauwkeurige weerspiegeling van het absolute aantal T-cellen, en niet alleen hun frequenties, wat misleidend kan zijn.Deze test maakt het...

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

Antigen Specific In Vivo Killing Assay using CFSE Labeled Target Cells

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

Antigen Specific In Vivo Killing Assay using CFSE Labeled Target Cells

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

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

Real Time Detection of In Vitro Tumor Cell Apoptosis Induced by CD8+ T Cells to Study Immune Suppressive Functions of Tumor-infiltrating Myeloid Cells

Potentiation of the tumor-killing ability of CD8+ T cells in tumors, along with their efficient tumor infiltration, is a key element of successful immunotherapies. Several studies have indicated that tumor infiltrating myeloid cells (e.g., myeloid-derived suppressor cells (MDSCs) and tumor-associated macrophages (TAMs)) suppress cytotoxicity of CD8+ T cells in the tumor microenvironment, and that targeting these regulatory myeloid cells can improve immunotherapies. Here, we present an in vitro assay system to evaluate immune suppressive effects of monocytic-MDSCs and TAMs on the tumor-killing ability of CD8+ T cells. To this end, we first cultured na&#239;ve splenic CD8+ T cells with anti-CD3/CD28 activating antibodies in the presence or absence of suppressor cells, and then co-cultured the pre-activated T cells with target cancer cells in the presence of a fluorogenic caspase-3 substrate. Fluorescence from the substrate in cancer cells was detected by real-time fluorescence microscopy as an indicator of T-cell induced tumor cell apoptosis. In this assay, we can successfully detect the increase of tumor cell apoptosis by CD8+ T cells and its suppression by pre-culture with TAMs or MDSCs. This functional assay is useful for investigating CD8+ T cell suppression mechanisms by regulatory myeloid cells and identifying druggable targets to overcome it via high throughput screening.

Real Time Detection of In Vitro Tumor Cell Apoptosis Induced by CD8+ T Cells to Study Immune Suppressive Functions of Tumor-infiltrating Myeloid Cells

We describe here a protocol to investigate cytotoxicity of pre-activated CD8+ T cells against cancer cells by detecting apoptotic cancer cells via real-time microscopy. This protocol can investigate mechanisms behind myeloid cell-induced T cell suppression and evaluate compounds aimed at replenishing T cells via blockade of immune suppressive myeloid cells.

Potentiation of the tumor-killing ability of CD8+ T cells in tumors, along with their efficient tumor infiltration, is a key element of successful ...

Cancer Research

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

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

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

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

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

Tumor Transplantation for Assessing the Dynamics of Tumor-Infiltrating CD8+ T Cells in Mice

T cell-mediated immunity plays a crucial role in immune responses against tumors, with cytotoxic T lymphocytes (CTLs) playing the leading role in eradicating cancerous cells. However, the origins and replenishment of tumor antigen-specific CD8+ T cells within the tumor microenvironment (TME) remain obscure. This protocol employs the B16F10-OVA melanoma cell line, which stably expresses the surrogate neoantigen, ovalbumin (OVA), and TCR transgenic OT-I mice, in which over 90% of CD8+ T cells specifically recognize the OVA-derived peptide OVA257-264 (SIINFEKL) bound to the class I major histocompatibility complex (MHC) molecule H2-Kb. These features enable the study of antigen-specific T cell responses during tumorigenesis.
Combining this model with tumor transplantation surgery, tumor tissues from donors were transplanted into tumor-matched syngeneic recipient mice to precisely trace the influx of recipient-derived immune cells into transplanted donor tissues, allowing the analysis of the immune responses of tumor-inherent and periphery-originated antigen-specific CD8+ T cells. A dynamic transition was found to occur between these two populations. Collectively, this experimental design has provided another approach to precisely investigate the immune responses of CD8+ T cells in TME, which will shed new light on tumor immunology.

Tumor Transplantation for Assessing the Dynamics of Tumor-Infiltrating CD8+ T Cells in Mice

Here, we present a tumor transplantation protocol for the characterization of tumor-inherent and periphery-derived tumor-infiltrated lymphocytes in a mouse tumor model. Specific tracing of the influx of recipient-derived immune cells with flow cytometry reveals the dynamics of the phenotypic and functional changes of these cells during antitumor immune responses.

T cell-mediated immunity plays a crucial role in immune responses against tumors, with cytotoxic T lymphocytes (CTLs) playing the leading role in ...

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 ...

Analysis of Human T Cell Activity in an Allogeneic Co-Culture Setting of Pre-Treated Tumor Cells

Cytotoxic T cells play a key role in the elimination of tumor cells and are, therefore, intensively studied in cancer immunology. The frequency and activity of cytotoxic T cells within tumors and their tumor microenvironment (TME) are now well-established prognostic and predictive biomarkers for numerous tumor types. However, it is well-known that various tumor treatment modalities, including radiotherapy, chemotherapy, immunotherapy, and targeted therapy, modulate not only the immunogenicity of the tumor but also the immune system itself. Consequently, the interaction between tumor cells and T cells requires more intensive study in different therapeutic contexts to fully understand the complex role of T cells during tumor therapy. To address this need, a protocol was developed to analyze the activity and proliferative capacity of human cytotoxic (CD8+) T cells in co-culture with pre-treated tumor cells. Specifically, CD8+ T cells from healthy donors are stained with the non-toxic proliferation marker carboxyfluorescein diacetate succinimidyl ester (CFSE) and stimulated using CD3/CD28-coated plates. Subsequently, T cells are co-cultured with pre-treated tumor cells. As a readout, T cell proliferation is quantified by measuring CFSE signal distribution and assessing the expression of surface activation markers via flow cytometry. This can be further complemented by quantifying cytokine release using enzyme-linked immunosorbent assay (ELISA). This method facilitates the evaluation of treatment-induced changes in the interaction between tumor cells and T cells, providing a foundation for more detailed analyses of tumor treatment modalities and their immunogenicity in a human ex vivo setting. Additionally, it contributes to the reduction of pre-clinical in vivo analyses.

The present protocol describes an experimental workflow that allows for the ex vivo analysis of human T cell stimulation in an allogenic co-culture system with pre-treated tumor cells.

Cytotoxic T cells play a key role in the elimination of tumor cells and are, therefore, intensively studied in cancer immunology. The frequency and ...

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.

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.

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

An In Vivo Mouse Model to Measure Na&#239;ve CD4 T Cell Activation, Proliferation and Th1 Differentiation Induced by Bone Marrow-derived Dendritic Cells

Quantification of na&#239;ve CD4 T cell activation, proliferation, and differentiation to T helper 1 (Th1) cells is a useful way to assess the role played by T cells in an immune response. This protocol describes the in vitro differentiation of bone marrow (BM) progenitors to obtain granulocyte macrophage colony-stimulating factor (GM-CSF) derived-dendritic cells (DCs). The protocol also describes the adoptive transfer of ovalbumin peptide (OVAp)-loaded GM-CSF-derived DCs and na&#239;ve CD4 T cells from OTII transgenic mice in order to analyze the in vivo activation, proliferation, and Th1 differentiation of the transferred CD4 T cells. This protocol circumvents the limitation of purely in vivo methods imposed by the inability to specifically manipulate or select the studied cell population. Moreover, this protocol allows studies in an in vivo environment, thus avoiding alterations to functional factors that may occur in vitro and including the influence of cell types and other factors only found in intact organs. The protocol is a useful tool for generating changes in DCs and T cells that modify adaptive immune responses, potentially providing important results to understand the origin or development of numerous immune associated diseases.

Here, we present a protocol for the in vivo determination of na&#239;ve CD4 T cell (T cell) activation, proliferation, and Th1 differentiation induced by GM-CSF bone marrow (BM)-derived dendritic cells (DCs). In addition, this protocol describes BM and T-cell isolation, DC generation, and DC and T-cell adoptive transfer.

Quantification of na&#239;ve CD4 T cell activation, proliferation, and differentiation to T helper 1 (Th1) cells is a useful way to assess the role played ...

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

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

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

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

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

Medicine

An Assay to Evaluate Immunodominance in Cytotoxic T Lymphocyte Responses

Source: Choi, J. et al., <a href="https://app.jove.com/v/59531">Tailoring In Vivo Cytotoxicity Assays to Study Immunodominance in Tumor-specific CD8+ T Cell Responses.</a> J. Vis. Exp. (2019)
The video showcases a flow cytometry-based assay for assaying immunodominance in cytotoxic T lymphocytes or CTL responses. Injected fluorescent target cells exhibit varying fluorescence corresponding to immunogenic epitopes. Post-spleen collection and sorting reveal a reduction in high-intensity fluorescent cells, confirming immunodominance in CTL responses.

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
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Tailoring In Vivo Cytotoxicity Assays to Study Immunodominance in Tumor-specific CD8⁺ T Cell Responses

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