BACKGROUNDQuantification 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.METHODSWe 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.RESULTSSIV 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.CONCLUSIONOur data support the concept that the GrB and IFN-gamma ELISPOT assays measure immune responses in different immune-cell populations with unique specificities.