O uso de interferão-γ Enzyme-linked Assay immunospot Caracterizar Novel epitopos de células T de papilomavírus humano

Summary

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.

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 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- 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 10⁵ cells per well typically used in ELISPOT assays^1,3, 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-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 epitopes⁴.

Protocol

By performing an IFN-γ 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-γ secretion prior to starting this protocol (Fig. 1). 1. Limiting Dilution of Epitope-Specific T Cells Prepare the feeder cell mixture by combining irradiated (4,000 rad) allogeneic peripheral bloo…

Discussion

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…

Materials

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