How to "spot" a real killer.

The Review by Otto O. Yang, ‘Will we be able to ‘spot’ an effective HIV vaccine?’, in the February issue of Trends in Immunology provides readers with a concise summary of the benefits and the caveats of current assays for detecting HIV-specific immune responses in infected patients [1xWill we be able to ‘spot’ an effective HIV-1 vaccine?. Yang, O.O. Trends Immunol. 2003; 24: 67–72Abstract | Full Text | Full Text PDF | PubMed | Scopus (64)See all References][1]. Both the interferon-γ (IFN-γ) ELISpot and tetramer staining provide valuable information about the frequency of cytokine-secreting T cells specific for a particular HIV epitope. However, the virus persists in infected individuals despite high frequencies of HIV-specific CD8 T cells detected by these assays. This leads Yang to speculate that it could be the use of synthetic peptides in these assays that is misleading because the representation of different peptides on non-infected cells pulsed with peptide mixtures might be quite different from those on HIV-infected cells. Thus, infection of targets with HIV-1 might be a more precise method of quantifying ‘effective’ immunity to HIV.In addition to the importance of defining the true protective epitopes, as explained by Yang, there is increasing suspicion that variations in the effector function of anti-HIV CD8 T cells might also contribute to antiviral efficacy. The cytolytic function of CD8 T cells is essential for the clearance of some viral infections [2xEffector CD4+ and CD8+ T-cell mechanisms in the control of respiratory virus infections. Doherty, P.C. et al. Immunol. Rev. 1997; 159: 105–117Crossref | PubMedSee all References][2] but until now there has been no method available that measures either the frequencies of cytolytic cells or the presence of small numbers of cytotoxic T lymphocytes (CTLs) within a large population of lymphocytes. We have recently developed such a method, the Lysispot, which will enable the evaluation of the frequencies of HIV-specific cytolytic cells directly ex vivo from the peripheral blood of infected patients. In the Lysispot assay [3xMeasuring the frequency of mouse and human cytotoxic T cells by the Lysispot assay: independent regulation of cytokine secretion and short-term killing. Snyder, J.E. et al. Nat. Med. 2003; 9: 231–236Crossref | PubMed | Scopus (65)See all References][3], target cells are ‘loaded’ with a foreign protein, β-galactosidase (β-gal), through transduction with Herpes Simplex virus (HSV)-Lac amplicons. These targets are then pulsed with peptide and added to ELISpot wells that are coated with an antibody specific for β-gal. Effector T-cell populations are added, and when a specific CTL meets and lyses a target cell, it causes the release of β-gal, which is captured in a localized area on the ELISpot filter. The cells are then washed away and the spots visualized using biotin-labeled anti-β-gal antibody as in a normal ELISpot. This method has revealed that the use of IFN-γ ELISpots to measure ‘CTL’ numbers can be misleading because the ratio of IFN-γ secretion to lysis can vary widely. We have also combined the Lysispot with the IFN-γ ELISpot in a two color fluorescent assay to detect individual T cells that only kill targets, only secrete IFN-γ, or exert both functions simultaneously. In addition to the importance of focusing on genuinely protective epitopes, as explained by Yang, we believe that it is crucial to define the actual effector functions that determine protective immunity. The Lysispot should be very useful for this purpose, supplementing other methods to provide a more complete picture of anti-HIV immunity.