title: Use of interferon-γ enzyme-linked immunospot assay to characterize novel T-cell epitopes of human papillomavirus.
abstract: 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-γ (IFN-γ) 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 collected(1). 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- γ secretion using commercially available magnetic beads (CD8 T-cell isolation kit, Miltenyi Biotec, Auburn CA). The selected IFN-γ 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- γ 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 10(5) cells per well typically used in ELISPOT assays(1,3), 1,000 T-cell clone cells in the presence of 1 x 10(5) 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-γ. This assures successful performance of IFN-γ ELISPOT assay. Similarly, IFN- γ 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- γ 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 epitopes(4).
authors.affiliation: Department of Microbiology and Parasitology, College of Basic Medical Sciences, China Medical University.
authors.forename: Xuelian
authors.initials: X
authors.lastname: Wang
authors.forename: William W
authors.initials: WW
authors.lastname: Greenfield
authors.forename: Hannah N
authors.initials: HN
authors.lastname: Coleman
authors.forename: Lindsey E
authors.initials: LE
authors.lastname: James
authors.forename: Mayumi
authors.initials: M
authors.lastname: Nakagawa
mesh_terms: D018414:CD8-Positive T-Lymphocytes / Q000276:immunology; D002460:Cell Line; D058501:Enzyme-Linked Immunospot Assay / Q000379:methods*; D018984:Epitopes, T-Lymphocyte / Q000032:analysis* / Q000276:immunology; D052162:Human papillomavirus 16 / Q000276:immunology*; D006801:Humans; D007371:Interferon-gamma / Q000276:immunology*
chemical_list: D018984:Epitopes, T-Lymphocyte; D007371:Interferon-gamma