This study was supported by an American Cancer Society Scholars Award (RSG-06-180-01-MBC) and NIH grants (R01 CA143130 and UL1RR029884).

By performing an IFN-&gamma; ELISPOT assay with a CD8 T cell line established from Subject 1, HPV 16 E6 46-70 region was determined to contain a T-cell epitope1 (Fig. 2), and the epitope-specific T cells were selected on the basis of IFN-&gamma; secretion prior to starting this protocol (Fig. 1).
1. Limiting Dilution of Epitope-Specific T Cells
<ol>
	<li>Prepare the feeder cell mixture by combining irradiated (4,000 rad) allogeneic peripheral bloo...

<ol>
	<li>Nakagawa, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+favorable+clinical+trend+is+associated+with+CD8+T-cell+immune+responses+to+the+human+papillomavirus+type+16+e6+antigens+in+women+being+studied+for+abnormal+pap+smear+results.">A favorable clinical trend is associated with CD8 T-cell immune responses to the human papillomavirus type 16 e6 antigens in women being studied for abnormal pap smear results.</a> J. Low. Genit. Tract Dis. 14, 124-129 (2010).</li><li>Walls, E., Crawford, L., Klaus, G. G. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Lymphocytes: A practical approach. , 149-149 (1987).</li><li>Nakagawa, M., Kim, K. H., Moscicki, A. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patterns+of+CD8+T-cell+epitopes+within+the+human+papillomavirus+type+16+(HPV+16)+E6+protein+among+young+women+whose+HPV+16+infection+has+become+undetectable.">Patterns of CD8 T-cell epitopes within the human papillomavirus type 16 (HPV 16) E6 protein among young women whose HPV 16 infection has become undetectable.</a> Clin. Diagn. Lab Immunol. 12, 1003-1005 (2005).</li><li>Wang, X., Santin, A. D., Bellone, S., Gupta, S., Nakagawa, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+CD4+T-cell+epitope+described+from+one+of+the+cervical+cancer+patients+vaccinated+with+HPV+16+or+18+E7-pulsed+dendritic+cells.">A novel CD4 T-cell epitope described from one of the cervical cancer patients vaccinated with HPV 16 or 18 E7-pulsed dendritic cells.</a> Cancer Immunol. Immunother. 58, 301-308 (2009).</li><li>Larsson, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+recombinant+vaccinia+virus+based+ELISPOT+assay+detects+high+frequencies+of+Pol-specific+CD8+T+cells+in+HIV-1-positive+individuals.">A recombinant vaccinia virus based ELISPOT assay detects high frequencies of Pol-specific CD8 T cells in HIV-1-positive individuals.</a> AIDS. 13, 767-777 (1999).</li><li>Nakagawa, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytotoxic+T+lymphocyte+responses+to+E6+and+E7+proteins+of+human+papillomavirus+type+16:+relationship+to+cervical+intraepithelial+neoplasia.">Cytotoxic T lymphocyte responses to E6 and E7 proteins of human papillomavirus type 16: relationship to cervical intraepithelial neoplasia.</a> J. Infect. Dis. 175, 927-931 (1997).</li><li>Wang, X., Moscicki, A. B., Tsang, L., Brockman, A., Nakagawa, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Memory+T+cells+specific+for+novel+human+papillomavirus+type+16+(HPV16)+E6+epitopes+in+women+whose+HPV16+infection+has+become+undetectable.">Memory T cells specific for novel human papillomavirus type 16 (HPV16) E6 epitopes in women whose HPV16 infection has become undetectable.</a> Clin. Vaccine Immunol. 15, 937-945 (2008).</li><li>Evans, 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+processing+defects+in+cervical+carcinomas+limit+the+presentation+of+a+CTL+epitope+from+human+papillomavirus+16+E6.">Antigen processing defects in cervical carcinomas limit the presentation of a CTL epitope from human papillomavirus 16 E6.</a> J. Immunol. 167, 5420-5428 (2001).</li><li>Shi, Y., Smith, K. D., Kurilla, M. G., Lutz, C. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytotoxic+CD8++T+cells+recognize+EBV+antigen+but+poorly+kill+autologous+EBV-infected+B+lymphoblasts:+immunodominance+is+elicited+by+a+peptide+epitope+that+is+presented+at+low+levels+in+vitro.">Cytotoxic CD8+ T cells recognize EBV antigen but poorly kill autologous EBV-infected B lymphoblasts: immunodominance is elicited by a peptide epitope that is presented at low levels in vitro.</a> J. Immunol. 159, 1844-1852 (1997).</li><li>Varadarajan, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+high-throughput+single-cell+analysis+of+human+CD8++T+cell+functions+reveals+discordance+for+cytokine+secretion+and+cytolysis.">A high-throughput single-cell analysis of human CD8+ T cell functions reveals discordance for cytokine secretion and cytolysis.</a> The Journal of Clinical Investigation. , (2011).</li><li>Nakagawa, M., Kim, K. H., Moscicki, A. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Different+methods+of+identifying+new+antigenic+epitopes+of+human+papillomavirus+type+16+E6+and+E7+proteins.">Different methods of identifying new antigenic epitopes of human papillomavirus type 16 E6 and E7 proteins.</a> Clin. Diagn. Lab Immunol. 11, 889-896 (2004).</li><li>Nakagawa, M., Kim, K. H., Gillam, T. M., Moscicki, A. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HLA+class+I+binding+promiscuity+of+the+CD8+T-cell+epitopes+of+human+papillomavirus+type+16+E6+protein.">HLA class I binding promiscuity of the CD8 T-cell epitopes of human papillomavirus type 16 E6 protein.</a> J. Virol. 81, 1412-1423 (2007).</li><li>Brown, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=T+cell+epitope+"hotspots"+on+the+HIV+Type+1+gp120+envelope+protein+overlap+with+tryptic+fragments+displayed+by+mass+spectrometry.">T cell epitope "hotspots" on the HIV Type 1 gp120 envelope protein overlap with tryptic fragments displayed by mass spectrometry.</a> AIDS Res. Hum. Retroviruses. 21, 165-170 (2005).</li><li>Masemola, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+and+promiscuous+CTL+epitopes+in+conserved+regions+of+Gag+targeted+by+individuals+with+early+subtype+C+HIV+type+1+infection+from+southern+Africa.">Novel and promiscuous CTL epitopes in conserved regions of Gag targeted by individuals with early subtype C HIV type 1 infection from southern Africa.</a> J. Immunol. 173, 4607-4617 (2004).</li><li>Walker, B. D., Korber, B. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immune+control+of+HIV:+the+obstacles+of+HLA+and+viral+diversity.">Immune control of HIV: the obstacles of HLA and viral diversity.</a> Nat. Immunol. 2, 473-475 (2001).</li></ol>

Traditionally, immunologists attempted to characterize novel T-cell epitopes by stimulating the T-cells of interest in vitro, and performing a limiting dilution experiment to isolate the epitope-specific T-cell clones. Then the T-cell clones were characterized using a chromium release assay 8,9. However, many attempts were unsuccessful because the frequencies of the T-cells may have been too low to be isolated or because not enough T-cell clone cells were available to complete the characterization. Th...

<table border="1">
<tbody>
 <tr>
 <td>Name of reagent</td>
 <td>Company</td>
 <td>Catalog Number</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td>CD8 T cell isolation kit</td>
 <td>Miltenyi</td>
 <td>130-094-156</td>
 <td></td>
 </tr>
 <tr>
 <td>Phytohemagglutinin</td>
 <td>Remel</td>
 <td>R-30852801</td>
 <td>2mg/vial</td>
 </tr>
 <tr>
 <td>Yssel's medium</td>
 <td>Gemini Bio-Products</td>
 <td>400-102</td>
 <td>1% pooled human serum</td>
 </tr>
 <tr>
 <td>96-well round bottom plate</td>
 <td>BD Falcon</td>
 <td>08-772-17</td>
 <td></td>
 </tr>
 <tr>
 <td>Recombinant human IL-2</td>
 <td>R&amp;D</td>
 <td>202-IL-050</td>
 <td>50&mu;g</td>
 </tr>
 <tr>
 <td>24-well plate</td>
 <td>Corning Costar</td>
 <td>07-200-84</td>
 <td></td>
 </tr>
 <tr>
 <td>Phosphate-buffered saline</td>
 <td>Cellgro</td>
 <td>MT-21-031-CV</td>
 <td></td>
 </tr>
 <tr>
 <td>Primary anti-IFN-&gamma; monoclonal antibody (D1K)</td>
 <td>Mabtech</td>
 <td>3420-3-1000</td>
 <td></td>
 </tr>
 <tr>
 <td>Biotin-conjugated anti-IFN-&gamma; monoclonal antibody (7B-6)</td>
 <td>Mabtech</td>
 <td>3420-6-1000</td>
 <td></td>
 </tr>
 <tr>
 <td>Multiscreen HA ELISPOT plate</td>
 <td>Millipore</td>
 <td>MAHA S45 10</td>
 <td></td>
 </tr>
 <tr>
 <td>Dimethyl Sulphoxide</td>
 <td>Sigma</td>
 <td>D2650</td>
 <td>100 ml bottle</td>
 </tr>
 <tr>
 <td>Pooled Serum, human</td>
 <td>Atlanta Biologicals</td>
 <td>S40510H</td>
 <td>Heat inactivate</td>
 </tr>
 <tr>
 <td>RPMI 1640</td>
 <td>Cellgro</td>
 <td>MT-10-040-CV</td>
 <td></td>
 </tr>
 <tr>
 <td>Tween-20</td>
 <td>Sigma</td>
 <td>P2287-100ml</td>
 <td></td>
 </tr>
 <tr>
 <td>Vectastain Elite ABC Kit</td>
 <td>Vector Labs</td>
 <td>NC9313719</td>
 <td></td>
 </tr>
 <tr>
 <td>Stable diaminobenzidine</td>
 <td>Open Biosystems</td>
 <td>MBI 1241</td>
 <td></td>
 </tr>
 <tr>
 <td>AID EliSpot Reader Classic</td>
 <td>Autoimmun Diagnostika GmbH</td>
 <td>ELR06</td>
 <td></td>
 </tr>
 </tbody>
</table>

use of interferon-&gamma; enzyme-linked immunospot assay to characterize novel t-cell epitopes of human papillomavirus

A protocol has been developed to overcome the difficulties of isolating and characterizing rare T cells specific for pathogens, such as human papillomavirus (HPV), that cause localized infections. The steps involved are identifying region(s) of HPV proteins that contain T-cell epitope(s) from a subject, selecting for the peptide-specific T cells based on interferon-&gamma; (IFN-&gamma;) secretion, and growing and characterizing the T-cell clones (Fig. 1). Subject 1 was a patient who was recently diagnosed with a high-grade squamous intraepithelial lesion by biopsy and underwent loop electrical excision procedure for treatment on the day the T cells were collected1. A region within the human papillomavirus type 16 (HPV 16) E6 and E7 proteins which contained a T-cell epitope was identified using an IFN- g enzyme-linked immunospot (ELISPOT) assay performed with overlapping synthetic peptides (Fig. 2). The data from this assay were used not only to identify a region containing a T-cell epitope, but also to estimate the number of epitope specific T cells and to isolate them on the basis of IFN- &gamma; secretion using commercially available magnetic beads (CD8 T-cell isolation kit, Miltenyi Biotec, Auburn CA). The selected IFN-&gamma; secreting T cells were diluted and grown singly in the presence of an irradiated feeder cell mixture in order to support the growth of a single T-cell per well. These T-cell clones were screened using an IFN- &gamma; ELISPOT assay in the presence of peptides covering the identified region and autologous Epstein-Barr virus transformed B-lymphoblastoid cells (LCLs, obtained how described by Walls and Crawford)2 in order to minimize the number of T-cell clone cells needed. Instead of using 1 x 105 cells per well typically used in ELISPOT assays1,3, 1,000 T-cell clone cells in the presence of 1 x 105 autologous LCLs were used, dramatically reducing the number of T-cell clone cells needed. The autologous LCLs served not only to present peptide antigens to the T-cell clone cells, but also to keep a high cell density in the wells allowing the epitope-specific T-cell clone cells to secrete IFN-&gamma;. This assures successful performance of IFN-&gamma; ELISPOT assay. Similarly, IFN- &gamma; ELISPOT assays were utilized to characterize the minimal and optimal amino acid sequence of the CD8 T-cell epitope (HPV 16 E6 52-61 FAFRDLCIVY) and its HLA class I restriction element (B58). The IFN- &gamma; ELISPOT assay was also performed using autologous LCLs infected with vaccinia virus expressing HPV 16 E6 or E7 protein. The result demonstrated that the E6 T-cell epitope was endogenously processed. The cross-recognition of homologous T-cell epitope of other high-risk HPV types was shown. This method can also be used to describe CD4 T-cell epitopes4.

Watch this Scientific Journal Video about Use of Interferon-Î³ Enzyme-linked Immunospot Assay to Characterize Novel T-cell Epitopes of Human Papillomavirus at JoVE.com

Use of Interferon-&amp;#947; Enzyme-linked Immunospot Assay to Characterize Novel T-cell Epitopes of Human Papillomavirus

Characterizing T-cell epitopes of pathogens that cause localized infections such as human papillomavirus is a challenge because of limited number of T cells in circulation. A method is described in which rare T cells were isolated and were characterized starting with a very small number of cells.

Use of Interferon-&gamma; Enzyme-linked Immunospot Assay to Characterize Novel T-cell Epitopes of Human Papillomavirus

A protocol has been developed to overcome the difficulties of isolating and characterizing rare T cells specific for pathogens, such as human ...

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Immunology and Infection

12.3K vistas.  China Medical University. Characterizing T-cell epitopes of pathogens that cause localized infections such as human papillomavirus is a challenge because of limited number of T cells in circulation. A method is described in which rare T cells were isolated and were characterized starting with a very small number of cells.