Chronic infection with Toxoplasma gondii can reactivate and cause life-threatening toxoplasmic encephalitis in AIDS patients and other immunocompromised individuals. The basis of persistence of this chronic infection is the tissue cyst. Thus, eliminating T. gondii cysts from chronically infected individuals before they become severely immunosuppressed will be able to prevent this serious disease. However, there are currently no drugs effective against the cysts. Our recent studies using a murine model uncovered that CD8+ immune T cells have a potent activity to directly penetrate into the cysts and induce their elimination through perforin- mediated mechanisms. We also identified that the N-terminal region (amino acids 41-152) of dense granule protein (GRA) 6 (GRA6Nt) of the parasite presented by the H-2Ld molecule is a key antigen to activate the anti- cyst CD8+ T cells. Our studies also identified that Iba1+ microglia and Ly6C+ macrophages accumulate to the cysts during the T cell-initiated immune process, and that these phagocytes kill T. gondii bradyzoites within the cysts, at least in part, by phagolysosome acidification. Based on these observations, the proposed studies are designed to obtain the information required for proceeding towards development of a vaccine to activate CD8+ T cells capable of targeting T. gondii cysts and eradicate chronic infection with this parasite in humans. For this purpose, we need to determine whether GRA6Nt activates anti-cyst CD8+ T cells through human MHC class I molecules, since the MHC class I molecules present antigens to activate CD8+ T cells.
Aim 1 will address this point by employing three transgenic mouse strains expressing either of the three major MHC class I supermotifs that cover 90% of human population. Once the anti-cyst T cells are primed, these T cells need to be recruited to cyst-containing cells and activated not only by recognizing T. gondii antigens but also by appropriate costimulatory molecules expressed on the cyst-containing cells. Our recent studies suggested an involvement of CXCL10 chemokine and inducible constimulator ligand (ICOSL) in these processes. Thus, Aim 2 will determine the roles of CXCL10 and ICOSL in recruitment and activation of anti-cyst CD8+ T cells, respectively. Another critical but unsolved issue is that small numbers of cysts persist in the brains of infected mice even in the presence of anti-cyst CD8+ T cells. Our recent study revealed that these persisting cysts express significantly greater levels of mRNA for selected GRA and rhoptry (ROP) proteins when compared to the overall cyst population that exists in the absence of the T cells. Since GRAs and ROPs are secreted from the parasite into infected cells, upregulated secretion of these selected GRAs and ROPs could play crucial roles in manipulating the functions of cyst-containing cells to prevent the attack by CD8+ T cells. Thus, Aim 3 focuses to determine whether these GRAs and ROPs play key roles in the evasion of the cysts from the T cell attack. We will also address the molecular mechanisms underlying this evasion process. The information from these studies will establish the basis for developing a novel method to eradicate chronic T. gondii infection.
Toxoplasma gondii establishes a chronic infection by forming tissue cysts preferentially in the brain, and reactivation of this chronic infection causes life-threatening toxoplasmic encephalitis in immunocompromised individuals such as those with AIDS. The proposed studies aim to identify T. gondii antigen applicable to a vaccine for activating the protective immunity capable of eliminating the cysts in humans and to uncover the mechanisms that T. gondii employs to escape from the protective immunity. The combination of the vaccine and a treatment to block the escape mechanism of the pathogen will allow us to develop a novel method to eradicate this widespread chronic infection in humans.
