Application of the granzyme B ELISPOT assay for monitoring cancer vaccine trials

J Immunother. May-Jun 2006;29(3):328-35. doi: 10.1097/01.cji.0000203079.35612.c8.


Granzyme B (GrB) is present in the granules of cytolytic lymphocytes and is a key mediator of cell-mediated target cell death via the granule-mediated pathway. The release of GrB can be used as an indicator of a cytotoxic T lymphocyte response. Herein, we report that the GrB enzyme-linked immunospot assay (ELISPOT) can be used to measure ex vivo antigen-specific cytotoxicity of peripheral blood mononuclear cells from cancer patients vaccinated with a peptide-based cancer vaccine. We compare the reactivity of patients' peripheral blood mononuclear cells in the GrB ELISPOT with reactivity in the tetramer, interferon (IFN)-gamma ELISPOT, and Cr-release assays. Differences in immune response over all assays tested were found between patients and 4 response patterns were observed. Reactivity in the GrB ELISPOT was more closely associated with cytotoxicity in the Cr-release assay than the tetramer or IFN-gamma ELISPOT assays. Moreover, the higher affinity g209-2M peptide (used for vaccination) elicited greater GrB secretion than the native g209 peptide, although this difference was not observed with IFN-gamma secretion. Taken together with the fact that GrB is a specific mediator released by cytotoxic T lymphocytes, these results show that simultaneous use of the GrB ELISPOT assay with other immunologic assays may provide important additional immunologic insight into patient responses to cancer vaccines.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Antigens / chemistry
  • Cancer Vaccines* / chemistry
  • Clinical Trials as Topic / methods*
  • Cytotoxicity Tests, Immunologic / methods
  • Enzyme-Linked Immunosorbent Assay / methods*
  • Granzymes
  • Humans
  • Immune System
  • Interferon-gamma / metabolism
  • Leukocytes, Mononuclear / metabolism
  • Melanoma / blood*
  • Melanoma / therapy
  • Membrane Glycoproteins / chemistry
  • Models, Statistical
  • Peptides / chemistry
  • Serine Endopeptidases / chemistry*
  • gp100 Melanoma Antigen


  • Antigens
  • Cancer Vaccines
  • Membrane Glycoproteins
  • PMEL protein, human
  • Peptides
  • g209-2M melanoma peptide
  • gp100 Melanoma Antigen
  • Interferon-gamma
  • GZMB protein, human
  • Granzymes
  • Serine Endopeptidases