CD8 + T-Cell Responses: Murine CTL Assays 437 437 From: Methods in Molecular Medicine, Vol. 72: Malaria Methods and Protocols Edited by: Denise L. Doolan © Humana Press, Inc., Totowa, NJ 38 Assessment of Antigen-Specific CTL- and CD8 + -Dependent IFN-γ Responses in Mice Katrin Peter, Régine Audran, Anilza Bonelo, Giampietro Corradin, and José Alejandro López 1. Introduction Mosquito injection of Plasmodium sporozoites into the bloodstream is followed by a short transit period before invasion of the hepatocyte, the only host cell expressing class I major histocompatibility complex (MHC) molecules. Liver stages develop within the hepatocyte for periods that vary according to the species, and they express various stage-specific proteins. Finally, schizonts are released into the bloodstream and invade the red blood cells, the final host cell before the continuation of the parasite cycle upon a subsequent mosquito bite. Protection to malaria could be efficiently obtained by immunization with irradiated sporozoites, a procedure that prevents parasite development upon repeated challenge with live sporozoites (1,2). Studies in human volunteers and animal models have shown that protection is dependent on both antibodies and T-cell responses (3–8). Among the targets for the immune response are the antigens expressed during the intrahepatic part of the life cycle, potentially presented in the context of MHC class I molecules to CD8 + lymphocytes. Moreover, CD8 + lymphocyte activity has been shown to be sufficient in protecting against disease in a mouse model (9). CD8 + lymphocyte activity is classically described as cytolytic, hence their designa- tion of cytolytic T lymphocytes (CTL). Specific CTL encounter target cells presenting antigen (peptides) by a MHC class I molecule and lyse them. This process is either the result of the release of molecules such as perforin or the activation of death signals, such as those mediated by CD95/Fas and the interaction with its ligand (10). This cy- tolytic function can be evaluated with the chromium release assay, an assay first devel- oped in 1968 by Brunner et al. (11), which continues to be the most specific detection and a sensitive system for the measurement of cytolytic activity. Additionally to the lytic activity, CTL also release interleukins upon antigen recog- nition; moreover, in the case of mouse malaria, the protective effect of CD8 + lympho- cytes appears to be mediated mainly through the release of interferon-γ (IFN-γ), and not through direct lysis of the infected cells. FAS and/or perforin-deficient mice are equally protected from Plasmodium berghei infection upon immunization with irradi- ated sporozoites (12). For this reason, the evaluation of the protective effect of CD8 +