Dissociated Production of Perforin, Granzyme B, and IFN-γ by HIV-Specific CD8⁺Cells in HIV Infection

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.

Resting of Cryopreserved PBMC Does Not Generally Benefit the Performance of Antigen-Specific T Cell ELISPOT Assays

“…Because the background was negligibly low for all PBMC samples when tested “fresh” or “rested”, in the graphical representation of the data for this paper we will omit the background spots. Such a wide range of recall response levels to individual antigens within different human donors is typically seen for non-cryopreserved PBMC ex vivo [14,15,18,20], even when donors are vaccinated at the same time and are tested at a given time point after vaccination [18]. It is even characteristic for recall responses of inbred mice when all parameters of the immunization, the genetic background and environmental influences are kept constant [4,7,9,17].…”

“…While initially IFN-γ ELISPOT assays found wide use for the detection of Th1/Tc1 cells, continued advancement in the ELISPOT field has enabled the detection of Th2/Tc2, Th17, cytolytic, and regulatory T cells via measurements of antigen-induced release of IL-2, IL-4, IL-5 [5,15,16], IL-17 [17], Granzyme B and perforin [18,19,20], and IL-10 [21], respectively. When it comes to the detection of polyfunctional T cells, dual color ELISPOT assays permit the measurement of cytokine coexpression in individual T cells with the same sensitivity as ICS, however, low frequency responses that are difficult to detect by ICS can be readily detected by dual color ELISPOT [22].…”

Lack of Disease Specificity Limits the Usefulness of In Vitro Costimulation in HIV- and HCV-Infected Patients

“…The intracellular presence of granzyme B or perforin, for example, merely indicates that the cell has encountered antigen within the past month [28]. In contrast, the actual release of granzyme B or perforin indicates immediate antigen recognition by a specific cell [1, 2]. …”

“…Particularly T cell responses have proven to be a valuable tool both for vaccine development and immune monitoring [1, 2]. Examining the phenotype and activation status of T cells (e.g., by FACS analysis) or their cytokine secretion profile (e.g., in ELISPOT assays) not only provides helpful information on the immunogenicity of individual viral antigens, but also reflects the patient's current state of immunocompetence, a vital piece of information for treatment and prognosis.…”

Human immunodeficiency virus type I-specific CD8+T cell subset abnormalities in chronic infection persist through effective antiretroviral therapy

“…This impairment manifests in phenotypic and functional changes affecting key CD8 + T cell properties such as proliferation, differentiation, cytokine production profile, signal transduction, and survival [1-8]. Compared to cytomegalovirus (CMV)-specific CD8 + T cells, HIV-specific CD8 + T cells are highly skewed towards effector memory (Tem) status, suggesting reduced differentiation into CD45RA + terminal effectors (Ttd) [9].…”