Summary

Het gebruik van interferon-γ Enzyme-linked immunospot Assay om nieuwe T-cel epitopen van het humaan papillomavirus karakteriseren

Published: March 08, 2012
doi:

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 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-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…

Disclosures

The authors have nothing to disclose.

Acknowledgements

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

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

References

  1. Nakagawa, M. 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. J. Low. Genit. Tract Dis. 14, 124-129 (2010).
  2. Walls, E., Crawford, L., Klaus, G. G. B. . Lymphocytes: A practical approach. , 149-149 (1987).
  3. Nakagawa, M., Kim, K. H., Moscicki, A. B. 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. Clin. Diagn. Lab Immunol. 12, 1003-1005 (2005).
  4. Wang, X., Santin, A. D., Bellone, S., Gupta, S., Nakagawa, M. A novel CD4 T-cell epitope described from one of the cervical cancer patients vaccinated with HPV 16 or 18 E7-pulsed dendritic cells. Cancer Immunol. Immunother. 58, 301-308 (2009).
  5. Larsson, M. A recombinant vaccinia virus based ELISPOT assay detects high frequencies of Pol-specific CD8 T cells in HIV-1-positive individuals. AIDS. 13, 767-777 (1999).
  6. Nakagawa, M. Cytotoxic T lymphocyte responses to E6 and E7 proteins of human papillomavirus type 16: relationship to cervical intraepithelial neoplasia. J. Infect. Dis. 175, 927-931 (1997).
  7. Wang, X., Moscicki, A. B., Tsang, L., Brockman, A., Nakagawa, M. Memory T cells specific for novel human papillomavirus type 16 (HPV16) E6 epitopes in women whose HPV16 infection has become undetectable. Clin. Vaccine Immunol. 15, 937-945 (2008).
  8. Evans, M. Antigen processing defects in cervical carcinomas limit the presentation of a CTL epitope from human papillomavirus 16 E6. J. Immunol. 167, 5420-5428 (2001).
  9. Shi, Y., Smith, K. D., Kurilla, M. G., Lutz, C. T. 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. J. Immunol. 159, 1844-1852 (1997).
  10. Varadarajan, N. A high-throughput single-cell analysis of human CD8+ T cell functions reveals discordance for cytokine secretion and cytolysis. The Journal of Clinical Investigation. , (2011).
  11. Nakagawa, M., Kim, K. H., Moscicki, A. B. Different methods of identifying new antigenic epitopes of human papillomavirus type 16 E6 and E7 proteins. Clin. Diagn. Lab Immunol. 11, 889-896 (2004).
  12. Nakagawa, M., Kim, K. H., Gillam, T. M., Moscicki, A. B. HLA class I binding promiscuity of the CD8 T-cell epitopes of human papillomavirus type 16 E6 protein. J. Virol. 81, 1412-1423 (2007).
  13. Brown, S. A. T cell epitope “hotspots” on the HIV Type 1 gp120 envelope protein overlap with tryptic fragments displayed by mass spectrometry. AIDS Res. Hum. Retroviruses. 21, 165-170 (2005).
  14. Masemola, A. M. Novel and promiscuous CTL epitopes in conserved regions of Gag targeted by individuals with early subtype C HIV type 1 infection from southern Africa. J. Immunol. 173, 4607-4617 (2004).
  15. Walker, B. D., Korber, B. T. Immune control of HIV: the obstacles of HLA and viral diversity. Nat. Immunol. 2, 473-475 (2001).

Play Video

Cite This Article
Wang, X., Greenfield, W. W., Coleman, H. N., James, L. E., Nakagawa, M. Use of Interferon-γ Enzyme-linked Immunospot Assay to Characterize Novel T-cell Epitopes of Human Papillomavirus. J. Vis. Exp. (61), e3657, doi:10.3791/3657 (2012).

View Video