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Updated: Aug 9, 2026
Analysis of Simian Immunodeficiency Virus-specific CD8+ T-cells in Rhesus Macaques by Peptide-MHC-I Tetramer Staining
Published on: December 23, 2016
Independence of granzyme B secretion and interferon- gamma production during acute simian immunodeficiency virus
Sandra A Calarota1, Miguel Otero, Tara M Robinson
1Department of Pathology and Laboratory Medicine, University of Pennsylvania School of Medicine, Philadelphia 19104-6100, USA.
Insights
Quantifying granzyme B (GrB) activity offers a new way to measure cytotoxic T lymphocyte (CTL) responses in SIV infection models. GrB ELISPOT assays capture distinct immune cell populations compared to interferon-gamma (IFN-gamma) ELISPOT assays.
Area of Science:
- Immunology
- Virology
- Primate Models
Background:
- Interferon-gamma (IFN-gamma) ELISPOT assays are standard for measuring cytotoxic T lymphocyte (CTL) activity in nonhuman primate models of simian immunodeficiency virus (SIV).
- However, IFN-gamma can be released by non-CTLs, potentially limiting its accuracy as a sole surrogate marker.
- Granzyme B (GrB), primarily secreted by activated CD8+ T cells, is proposed as an additional, more specific CTL activity marker.
Purpose of the Study:
- To evaluate granzyme B (GrB) activity as a surrogate marker for CTL responses in rhesus macaques infected with SIV.
- To compare GrB ELISPOT responses with IFN-gamma ELISPOT responses and plasma viral load during acute SIV infection.
Main Methods:
- Enzyme-linked immunospot (ELISPOT) assays were used to measure GrB activity in response to SIV Gag peptide pools.
- 18 rhesus macaques with acute SIVmac251 infection were studied.
- Correlations between GrB activity, IFN-gamma production, and plasma viral load were analyzed.
Main Results:
- SIV Gag-specific GrB activity significantly increased post-infection (3.9- to 14.4-fold).
- GrB secretion did not directly correlate with IFN-gamma production.
- IFN-gamma production negatively correlated with viral load, while GrB activity did not; GrB activity peaked when viral load was lowest.
Conclusions:
- GrB and IFN-gamma ELISPOT assays measure distinct immune responses.
- GrB ELISPOT assays may provide a more specific assessment of CTL activity in SIV-infected nonhuman primates.
- These findings highlight the utility of GrB as a complementary marker for CTL assessment in viral infection models.
Background:
Quantification of interferon (IFN)-gamma by enzyme-linked immunospot (ELISPOT) assay is currently used as a surrogate measurement of cytotoxic T lymphocyte (CTL) activity in nonhuman primates, particularly in simian immunodeficiency virus (SIV) models. Given that noncytotoxic cells and natural killer cells can also release IFN-gamma, quantification of granzyme B (GrB), a molecule secreted predominantly by activated CD8+ T cells, may represent an additional surrogate measurement of CTL activity.
Methods:
We evaluated, by ELISPOT assay, GrB activity in response to 3 overlapping SIV Gag peptide pools in 18 rhesus macaques with acute SIVmac251 infection and analyzed its correlation with IFN-gamma ELISPOT responses and plasma viral load.
Results:
SIV Gag-specific GrB activity increased from 3.9- to 14.4-fold after infection, compared with that observed before infection. GrB secretion did not correlate directly with IFN-gamma production. Importantly, SIV Gag-specific IFN- gamma production was negatively correlated with plasma viral load, whereas GrB activity was not. However, the peak of GrB activity coincided with the lowest plasma viral load detected after infection, whereas the magnitude of IFN-gamma production was 1.8-fold lower than the GrB response; these results illustrate that the responses differ.
Conclusion:
Our data support the concept that the GrB and IFN-gamma ELISPOT assays measure immune responses in different immune-cell populations with unique specificities.
