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Immunology and Infection

Användning av interferon-γ Enzymkopplad immunospot Analys att karaktärisera nya T-cellsepitoper av humant papillomvirus

Published: March 8, 2012 doi: 10.3791/3657

Materials

Name Company Catalog Number Comments
CD8 T cell isolation kit Miltenyi Biotec 130-094-156
Phytohemagglutinin Remel, Thermo Fisher Scientific R-30852801 2mg/vial
Yssel’s medium Gemini Bio Products 400-102 1% pooled human serum
96-well round bottom plate BD Biosciences 08-772-17
Recombinant human IL-2 R&D Systems 202-IL-050 50μg
24-well plate Corning 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 EMD Millipore MAHA S45 10
Dimethyl Sulphoxide Sigma-Aldrich D2650 100 ml bottle
Pooled Serum, human Atlanta Biologicals S40510H Heat inactivate
RPMI 1640 Cellgro MT-10-040-CV
Tween-20 Sigma-Aldrich P2287-100ml
Vectastain Elite ABC Kit Vector Laboratories NC9313719
Stable diaminobenzidine Open Biosystems MBI 1241
AID EliSpot Reader Classic Autoimmun Diagnostika GmbH ELR06

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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. Lymphocytes: A practical approach. Klaus, G. G. B. , IRL Press. 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).
Användning av interferon-γ Enzymkopplad immunospot Analys att karaktärisera nya T-cellsepitoper av humant papillomvirus
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Cite this Article

Wang, X., Greenfield, W. W.,More

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).

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