Independence of Granzyme B Secretion and Interferon‐γ Production during Acute Simian Immunodeficiency Virus Infection

William Penn University, Filadelfia, Pennsylvania, United States
The Journal of Infectious Diseases (Impact Factor: 6). 06/2006; 193(10):1441-50. DOI: 10.1086/503364
Source: PubMed


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

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    • "The breadth of the response differed between each animal with only the immunodominant Gag_CM9 (Allen et al., 1998) detected in all 5 macaques. CD8 + T cells that have cytolytic effector function may be critical for control of HIV/SIV replication (Calarota et al., 2006; Goulder et al., 2000; Yang et al., 1997), but IFN-γ-producing CD8 + T cell responses detected by the ELISPOT assay do not always correlate with the cytolytic effector response (Calarota et al., 2006). To determine if the HBcAg-multi-epitope DNA vaccine induced CD8 + T cells with cytolytic activity, a second group of macaques was immunized with 3 doses of HBc-multiepitope DNA vaccine without the plasmid expressing whole tat, and cytolytic effector responses were assayed by 51 Cr-release assay. "
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    ABSTRACT: An effective HIV vaccine will likely need to induce broad and potent CTL responses. Epitope-based vaccines offer significant potential for inducing multi-specific CTL, but often require conjugation to T helper epitopes or carrier moieties to induce significant responses. We tested hybrid DNA vaccines encoding one or more HIV or SIV CTL epitopes fused to a hepatitis B core antigen (HBcAg) carrier gene as a means to improve the immunogenicity of epitope-based DNA vaccines. Immunization of mice with a HBcAg-HIV epitope DNA vaccine induced CD8(+) T cell responses that significantly exceeded levels induced with DNA encoding either the whole HIV antigen or the epitope alone. In rhesus macaques, a multi-epitope hybrid HBcAg-SIV DNA vaccine induced CTL responses to 13 different epitopes, including 3 epitopes that were previously not detected in SIV-infected macaques. These data demonstrate that immunization with hybrid HBcAg-epitope DNA vaccines is an effective strategy to increase the magnitude and breadth of HIV-specific CTL responses.
    Full-text · Article · Sep 2007 · Virology
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    ABSTRACT: Flow-cytometric conditions for detection of lysosomal-associated membrane proteins (LAMPs) on the surface of recently degranulated cells were optimized for rhesus macaques and used to investigate the functional properties of rhesus cytomegalovirus (rhCMV)-specific CD8+ T lymphocytes with regards to cytotoxicity and interferon (IFN)-gamma secretion in six asymptomatic CMV-seropositive rhesus macaques. Unlike humans, the rhesus macaque LAMP-1 protein CD107a underwent little or no endocytosis over a six to 18 h stimulation period. Following in vitro stimulation, rhCMV-specific CD8+ T lymphocytes were heterogeneous with regards to the composition of cells positive for CD107a and/or IFN-gamma, time to reach peak degranulation, and kinetics of IFN-gamma secretion relative to degranulation. Responder CD8+ T lymphocytes that underwent degranulation without IFN-gamma production (CD107a+IFN-gamma-) were predominantly composed of terminally differentiated effectors (CD28-CD45RA+). Moreover, they had significantly lower frequencies of effector memory (CD28-CD45RA-) cells compared to the IFN-gamma-secreting cells that did or did not undergo degranulation (CD107a+IFN-gamma+ or CD107a-IFN-gamma+). The perforin content of effector CD8+ T lymphocytes was significantly greater than that of effector memory CD8+ T lymphocytes in rhesus macaques, suggesting that they were more cytolytic. Our findings suggest that the composition of rhCMV-specific CD8+ T lymphocytes with regards to CD107a+IFN-gamma- responders may be an important determinant of their ability to control CMV replication.
    Preview · Article · Sep 2007 · Journal of Immunological Methods
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    ABSTRACT: CD8(+) T cells play a crucial role in the control of viral infections such as HIV. The functional characterization of HIV-specific CD8(+) T cells has so far been largely restricted to studies of IFN-gamma. The TCR-triggered release of the effector molecules perforin (PFN) and granzyme B (GzB), however, is thought to be a central pathway for the destruction of virus-infected target cells by CD8(+) effector T cells. Here we would like to address two major findings. On the one hand we propose that ex vivo measurements of PFN and GzB secretion via ELISPOT may permit the distinction between in vivo resting versus activated CD8(+) memory T cells in healthy and HIV-infected individuals. Therefore, extending the present standard of IFN-gamma measurements to the analysis of PFN and GzB release in functional T cell assays will provide new insights into CD8(+) effector T cell functions. It should enable the evaluation of therapeutic vaccination efficacy by its ability to reactivate and convert IFN-gamma-positive, but GzB- and PFN-negative memory CD8(+) T cells into PFN/GzB-secreting effector cells. On the other hand, we report on a frequent ex vivo dissociation of the HIV peptide-induced secretion of PFN and GzB in chronic HIV infection underlining CD8(+) effector T cell diversity in this disease--an aspect that also has to be accounted for in immune monitoring approaches.
    No preview · Article · Feb 2008 · AIDS Research and Human Retroviruses
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