Rapid qualitative and quantitative analysis of T-cell responses in HIV-1-infected individuals receiving successful HAART and HIV-1 sero-negative controls: concomitant assessment of perforin, IFN-gamma and IL-4 secretion.

The enzyme-linked immunospot (ELIspot) assay is a highly sensitive and valuable tool for determining the frequency of cytokine-secreting T cells. It is essential to determine both frequencies and functional capabilities of antigen-specific T cells, including cytokine secretion, degranulation, and cytotoxicity in order to obtain a fuller picture of the immune status of an individual. We describe here for the first time a perforin-release ELIspot assay which, when used in combination with IFN-gamma and IL-4 ELIspots, permits rapid assessment of these functional parameters for antigen-specific T cells. Whole antigen or peptides from HIV-1, recall and other viral antigens were used for in vitro stimulation. Anti-HIV-1 responses in treated chronically infected individuals were weak, both in terms of perforin and IFN-gamma production. Tetanus toxoid stimulation was associated with moderate perforin release and a predominantly type-2 IL-4 producing response, whilst herpes simplex virus antigen stimulation resulted in perforin release but only a weak type-1 IFN-gamma response. Anti-cytomegalovirus responses generated high levels of perforin in conjunction with IFN-gamma. Cytokines IL-2 and IL-12/IL-15 induced perforin release coupled with an IFN-gamma type-1 response. Perforin release strongly correlated with IFN-gamma production to individual influenza, Epstein-Barr virus or cytomegalovirus MHC class I restricted peptides, in an HIV-1 sero-negative cohort, indicating a cytolytic type-1 CD8+ T-cell response. Evaluation of immunogenicity and putative efficacy of candidate vaccines using IFN-gamma will not be as informative alone as when combined with perforin and IL-4 evaluations, which allow assessment of specific cytotoxic potential without extensive cell culture.