Name: tailoring in vivo cytotoxicity assays to study immunodominance in tumor-specific cd8+ t cell responses
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Description: Watch this Scientific Journal Video about Tailoring In Vivo Cytotoxicity Assays to Study Immunodominance in Tumor-specific CD8+ T Cell Responses at JoVE.com

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
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	<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...

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J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cross-priming+induces+immunodomination+in+the+presence+of+viral+MHC+class+I+inhibition.">Cross-priming induces immunodomination in the presence of viral MHC class I inhibition.</a> PLoS Pathogens. 14 (2), e1006883 (2018).</li><li>Crowe, S. R., 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=Differential+antigen+presentation+regulates+the+changing+patterns+of+CD8++T+cell+immunodominance+in+primary+and+secondary+influenza+virus+infections.">Differential antigen presentation regulates the changing patterns of CD8+ T cell immunodominance in primary and secondary influenza virus infections.</a> The Journal of Experimental Medicine. 198 (3), 399-410 (2003).</li><li>Probst, H. C., 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=Immunodominance+of+an+antiviral+cytotoxic+T+cell+response+is+shaped+by+the+kinetics+of+viral+protein+expression.">Immunodominance of an antiviral cytotoxic T cell response is shaped by the kinetics of viral protein expression.</a> The Journal of Immunology. 171 (10), 5415-5422 (2003).</li><li>Gileadi, U., 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=Generation+of+an+immunodominant+CTL+epitope+is+affected+by+proteasome+subunit+composition+and+stability+of+the+antigenic+protein.">Generation of an immunodominant CTL epitope is affected by proteasome subunit composition and stability of the antigenic protein.</a> The Journal of Immunology. 163 (11), 6045-6052 (1999).</li><li>Zanker, D., Waithman, J., Yewdell, J. W., Chen, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mixed+proteasomes+function+to+increase+viral+peptide+diversity+and+broaden+antiviral+CD8++T+cell+responses.">Mixed proteasomes function to increase viral peptide diversity and broaden antiviral CD8+ T cell responses.</a> The Journal of Immunology. 191 (1), 52-59 (2013).</li><li>Deng, Y., Yewdell, J. W., Eisenlohr, L. C., Bennink, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MHC+affinity,+peptide+liberation,+T+cell+repertoire,+and+immunodominance+all+contribute+to+the+paucity+of+MHC+class+I-restricted+peptides+recognized+by+antiviral+CTL.">MHC affinity, peptide liberation, T cell repertoire, and immunodominance all contribute to the paucity of MHC class I-restricted peptides recognized by antiviral CTL.</a> The Journal of Immunology. 158 (4), 1507-1515 (1997).</li><li>Chen, W., Khilko, S., Fecondo, J., Margulies, D. H., McCluskey, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determinant+selection+of+major+histocompatibility+complex+class+I-restricted+antigenic+peptides+is+explained+by+class+I-peptide+affinity+and+is+strongly+influenced+by+nondominant+anchor+residues.">Determinant selection of major histocompatibility complex class I-restricted antigenic peptides is explained by class I-peptide affinity and is strongly influenced by nondominant anchor residues.</a> The Journal of Experimental Medicine. 180 (4), 1471-1483 (1994).</li><li>Kotturi, M. 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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=T+cells+compete+for+access+to+antigen-bearing+antigen-presenting+cells.">T cells compete for access to antigen-bearing antigen-presenting cells.</a> The Journal of Experimental Medicine. 192 (8), 1105-1113 (2000).</li><li>Kastenmuller, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cross-competition+of+CD8++T+cells+shapes+the+immunodominance+hierarchy+during+boost+vaccination.">Cross-competition of CD8+ T cells shapes the immunodominance hierarchy during boost vaccination.</a> The Journal of Experimental Medicine. 204 (9), 2187-2198 (2007).</li><li>Memarnejadian, A., 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=PD-1+Blockade+Promotes+Epitope+Spreading+in+Anticancer+CD8(+)+T+Cell+Responses+by+Preventing+Fratricidal+Death+of+Subdominant+Clones+To+Relieve+Immunodomination.">PD-1 Blockade Promotes Epitope Spreading in Anticancer CD8(+) T Cell Responses by Preventing Fratricidal Death of Subdominant Clones To Relieve Immunodomination.</a> The Journal of Immunology. 199 (9), 3348-3359 (2017).</li><li>Haeryfar, S. M., DiPaolo, R. J., Tscharke, D. C., Bennink, J. R., Yewdell, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulatory+T+cells+suppress+CD8++T+cell+responses+induced+by+direct+priming+and+cross-priming+and+moderate+immunodominance+disparities.">Regulatory T cells suppress CD8+ T cell responses induced by direct priming and cross-priming and moderate immunodominance disparities.</a> The Journal of Immunology. 174 (6), 3344-3351 (2005).</li><li>Rytelewski, 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=Suppression+of+immunodominant+antitumor+and+antiviral+CD8++T+cell+responses+by+indoleamine+2,3-dioxygenase.">Suppression of immunodominant antitumor and antiviral CD8+ T cell responses by indoleamine 2,3-dioxygenase.</a> PLoS One. 9 (2), e90439 (2014).</li><li>Maleki Vareki, 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=Differential+regulation+of+simultaneous+antitumor+and+alloreactive+CD8(+)+T-cell+responses+in+the+same+host+by+rapamycin.">Differential regulation of simultaneous antitumor and alloreactive CD8(+) T-cell responses in the same host by rapamycin.</a> American Journal of Transplantation. 12 (1), 233-239 (2012).</li><li>Irvine, K., Bennink, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Factors+influencing+immunodominance+hierarchies+in+TCD8++-mediated+antiviral+responses.">Factors influencing immunodominance hierarchies in TCD8+ -mediated antiviral responses.</a> Expert Review of Clinical Immunology. 2 (1), 135-147 (2006).</li><li>Grossmann, M. E., Davila, T., Celis, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Avoiding+tolerance+against+prostatic+antigens+with+subdominant+peptide+epitopes.">Avoiding tolerance against prostatic antigens with subdominant peptide epitopes.</a> Journal of Immunotherapy. 24 (3), 237-241 (2001).</li><li>Schreiber, H., Wu, T. H., Nachman, J., Kast, W. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunodominance+and+tumor+escape.">Immunodominance and tumor escape.</a> Seminars in Cancer Biology. 12 (1), 25-31 (2002).</li><li>Mylin, L. 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=Quantitation+of+CD8(+)+T-lymphocyte+responses+to+multiple+epitopes+from+simian+virus+40+(SV40)+large+T+antigen+in+C57BL/6+mice+immunized+with+SV40,+SV40+T-antigen-transformed+cells,+or+vaccinia+virus+recombinants+expressing+full-length+T+antigen+or+epitope+minigenes.">Quantitation of CD8(+) T-lymphocyte responses to multiple epitopes from simian virus 40 (SV40) large T antigen in C57BL/6 mice immunized with SV40, SV40 T-antigen-transformed cells, or vaccinia virus recombinants expressing full-length T antigen or epitope minigenes.</a> Journal of Virology. 74 (15), 6922-6934 (2000).</li><li>Fu, T. 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=An+endoplasmic+reticulum-targeting+signal+sequence+enhances+the+immunogenicity+of+an+immunorecessive+simian+virus+40+large+T+antigen+cytotoxic+T-lymphocyte+epitope.">An endoplasmic reticulum-targeting signal sequence enhances the immunogenicity of an immunorecessive simian virus 40 large T antigen cytotoxic T-lymphocyte epitope.</a> Journal of Virology. 72 (2), 1469-1481 (1998).</li><li>Chen, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cross-priming+of+CD8++T+cells+by+viral+and+tumor+antigens+is+a+robust+phenomenon.">Cross-priming of CD8+ T cells by viral and tumor antigens is a robust phenomenon.</a> European Journal of Immunology. 34 (1), 194-199 (2004).</li><li>Memarnejadian, A., Meilleur, C. E., Mazzuca, D. M., Welch, I. D., Haeryfar, S. 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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. 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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 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 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 ...

This assay can be employed to quantify T-cell-mediated cytotoxicity against two distinct peptide epitopes of tumor antigens simultaneously. The in vivo nature of this assay allows for cytotoxic function of T-cells to be measured within the intact architecture of a secondary lymphoid organ. In addition, the killing observed is an accurate reflection of the absolute number of T-cells, and not just their frequencies, which can be misleading.This assay enables the examination of cytotoxic T cell responses to immunodominant and sub-dominant tumor antigens. It therefore provides valuable information when you're designing a vaccine, as well as immunotherapeutic protocols for cancer. Our protocol has been tailored to study CD8 T cell responses.However, it can be modified to measure cytotoxicity elicited by other killer cell types, such as NK cells and innate-like T lymphocytes. This assay requires experience in aseptic technique, mouse tissue handling, target cell prep, as well as expertise in tail vein injections. One needs to acquire this skill set before attempting the in vivo killing assay.Once the T-antigen-positive adherent tumor cell line has become fully confluent or slightly over-confluent, gently rinse the monolayer with sterile, warm PBS, and detach the tumor cells with 0.25%trypsin EDTA in a biosafety cabinet. Tap the sides of the culture flask several times to release the remaining adherent cells, and stop the reaction after about five minutes with the addition of five milliliters of DMEM medium. Pipette the cell solution a few times before filtering the cell suspension through a 70-micrometer-pore cell strainer into a 50-milliliter conical tube.Collect the cells by centrifugation, and resuspend the pellet in 10 milliliters of sterile, cold PBS for the first of three washes under the same conditions. After the third wash, resuspend the cells at a four times 10 to the seven cells per milliliter of sterile PBS concentration, and inject 500 microliters of the T-antigen-positive tumor cell suspension intraperitoneally into each six-to-12-week-old C57 Black 6 mouse recipient. For target splenocyte preparation, place the euthanized donor mouse in the prone position, and spray the animal with 70%ethanol.Using sterile forceps and scissors, lift the skin and make a small ventral midline incision. Cut the skin in a cross-like fashion to expose the peritoneum, and use the forceps to pull up the peritoneum in a tent-like fashion, without catching any of the internal organs. Cut open the peritoneum to expose the peritoneal cavity, and gently transfer the spleen to a 15-milliliter Dounce tissue grinder containing five milliliters of sterile PBS.Use the plunger to apply manual pressure until the splenic tissue dissipates into a red homogeneous cell suspension, and transfer the homogenate into a 15-milliliter tube. Collect the suspension by centrifugation, and resuspend the pellet in four milliliters of ammonium chloride potassium lysine buffer to eliminate the erythrocytes. After four minutes, stop the process with eight milliliters of complete medium, and filter the cell suspension through a 70-micrometer-pore cell strainer into a new 50-milliliter tube.Separate the white blood cells from the red blood cell debris by centrifugation, and resuspend the pellet in 12 milliliters of complete medium. Then split the splenocyte suspension into three equal four-milliliter aliquots. For peptide coding of the target splenocytes, pulse one splenocyte aliquot with one micromolar of irrelevant peptide, one aliquot with one micromolar of Site I peptide, and one aliquot with one micromolar of Site IV peptide for one hour at 37 degrees Celsius and 5%carbon dioxide.At the end of the incubation, remove the excess peptide by centrifugation and wash the peptide-pulsed splenocytes two times in 12 milliliters of sterile, cold PBS per wash per tube. After the second wash, resuspend the cells in four milliliters of fresh, sterile PBS per tube. Add 025, 0.25, and two-micromolar CFSE into the irrelevant, Site I, and Site IV peptide-pulsed splenocyte suspensions respectively.Place the tubes at 37 degrees Celsius for 15 minutes with manual inversion once every five minutes. At the end of the incubation, add three milliliters of heat-inactivated fetal bovine serum to each tube to stop the CFSE reaction and bring the final volume in each tube up to 15 milliliters with PBS. Then collect the dye-labeled cells by centrifugation for two washes in 12 milliliters of fresh, sterile PBS per wash.To confirm an adequate CFSE labeling of the target splenocytes, resuspend the pellet in three milliliters of PBS and mix with gentle vortexing. Transfer 10 microliters from each CFSE-labeled cell suspension into an individual five-milliliter round bottom polystyrene fluorescence-activated cell sorting, or FACS tube, containing PBS, and load the tube onto a flow cytometer equipped with a 488-nanometer laser. In the flow cytometer software, create a lymphocyte gate based on the forward scatter and side scatter properties of the cells before acquiring 5, 000 events falling within the lymphocyte gate in the Fluorescence 1 channel.Within the parent CFSE-positive population, draw additional histogram gates to identify the CFSE-low, CFSE-intermediate, and CFSE-high subpopulations. Then confirm an equal or a near-equal event number within the three gates for the other two tubes of labeled cells. For injection of the target cells, first gently vortex the source tubes and pool the three CFSE-labeled cell suspensions into a new tube at equal ratios.Top up the tube contents with sterile PBS, and collect the cells by centrifugation for counting. Then adjust the volume to a one times 10 to the seven mixed target cells per 200 microliters of PBS per recipient concentration, and inject 200 microliter volumes of the cell suspension into the tail vein of each recipient C57 Black 6 mouse. Two or four hours after the injection, remove and process the spleen of each recipient animal as demonstrated, and resuspend the isolated white blood cells in three milliliters of PBS per spleen.Transfer approximately one times 10 to the seven cells from each processed spleen into a clean FACS tube, and immediately run the cells on the flow cytometer as demonstrated to gate the CFSE-low, intermediate, and high target cell populations. Then acquire a total of 2, 000 CFSE-low events in the Fluorescence 1 channel, and calculate the specific lysis of each cognate target cell population according to the formula detailed in the text. In this representative experiment, the success of the depletion of naturally occurring regulatory T-cells in mice injected with anti-CD25 monoclonal antibody was confirmed by flow cytometry.As expected, near-equal peaks corresponding to the control and cognate target cells were detectable in naive mice. In contrast, Site-IV-displaying target cells were almost completely absent in T-antigen-primed mice, regardless of their prior treatment with anti-CD25 monoclonal antibody or PBS. Interestingly, naturally occurring T reg cell depletion by anti-CD25 monoclonal antibody augmented in vivo cytotoxic T-lymphocyte-mediated lysis of Site-I-pulsed target cells.In this second representative investigation, programmed death 1 blockade enhanced sub-dominant CD8-positive T cell sites one and two-slash-three-specific responses, suggesting that interfering with programmed death 1/programmed death ligand 1 interactions may induce epitope-spreading in anti-cancer CD8-positive T cell responses. It's critical when labeling your donor cells with CFSE, make sure your concentrations are precise. Otherwise this may result in overlapping histogram peaks, which make your required calculations and data interpretation difficult, if not impossible.It will be important to optimize in vivo cytotoxicity assays to quantify effector functions elicited by different killer cell types that recognize peptide and non-peptide antigens in the same host.

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