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Clinical and Vaccine Immunology, April 2007, p. 362-368, Vol. 14, No. 4
1071-412X/07/$08.00+0     doi:10.1128/CVI.00460-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Detection of Human Papillomavirus Type 16-Specific T Lymphocytes by a Recombinant Vaccinia Virus-Based Enzyme-Linked Immunospot Assay{triangledown}

Kevin H. Kim,1* William Greenfield,2 Ezekiel Shotts,1 and Mayumi Nakagawa1,3

Departments of Dermatology,1 Obstetrics and Gynecology,2 Pathology, University of Arkansas for Medical Sciences, Little Rock, Arkansas3

Received 7 December 2006/ Returned for modification 22 January 2007/ Accepted 8 February 2007

Cell-mediated immunity, particularly that induced by T cells, is thought to have a key role in controlling infection. The enzyme-linked immunospot (ELISPOT) assay has been successfully adapted to detect T-cell immune response to a variety of pathogens. However, it still remains a challenge to detect antigen-specific T cells when the numbers of circulating cells are low, such as in a local cervical infection caused by genital human papillomavirus (HPV). The goal of this study was to develop a protocol for enhanced detection of HPV-specific CD8+ T cells by examining a number of the variables involved in performing an ELISPOT assay. Since blood samples consistently positive for HPV-specific T cells are difficult to obtain, previously described human papillomavirus type 16 (HPV16) E6 52-61 (FAFRDLCIVY)-specific T-cell clone cells (13) seeded in peripheral blood mononuclear cells from an HLA-B57-positive blood donor were used. The variables examined were the amounts of primary and secondary anti-gamma interferon antibodies, amounts of antigen-presenting monocytes and recombinant vaccinia virus expressing the HPV16 E6 protein, and amounts of exogenous cytokines added (recombinant human interleukin-2 [rhIL-2] and rhIL-7). The amounts of antigen-presenting monocytes, followed by the concentration of exogenous rhIL-2, had the most pronounced and significant effects in enhancing sensitivity of the ELISPOT assay. Blood samples from six patients being monitored for abnormal Pap smear results and from 12 healthy volunteers were examined using the enhanced conditions.

* Corresponding author. Mailing address: Department of Dermatology, School of Medicine, University of Arkansas for Medical Sciences, 4301 West Markham Street, Slot 576, Little Rock, AR 72205. Phone: (501) 686-8635. Fax: (501) 686-7264. E-mail: mnakagawa{at}uams.edu

{triangledown} Published ahead of print on 21 February 2007.

Clinical and Vaccine Immunology, April 2007, p. 362-368, Vol. 14, No. 4
1071-412X/07/$08.00+0     doi:10.1128/CVI.00460-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.





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