Final Report Summary - APATRIVAP (Antigen processing and T cell recognition of intravacuolar parasites) CD8 T lymphocytes play an essential role in protection against infectious agents such as intracellular parasites. CD8 T cells detect short peptides (8-10 amino acids) presented by Major Histocompatibility Complex (MHC) class I molecules on the surface of antigen-presenting cells (APCs). An important source for these peptides is the degradation products of self- or virus-encoded cytosolic proteins. But it is also recognized that proteins acquired from exogenous sources can be used for presentation by MHC I molecules. This pathway, typically referred to as “cross-presentation”, is key since it ensures detection of cells infected by pathogens residing in intracellular vacuoles, i.e. segregated from the cytosol.T. gondii is an obligate intravacuolar parasite which establishes chronic infection in a wide range of hosts, including humans. In humans, T. gondii can cause life-threatening encephalitis in the immunocompromised and lead to birth defects if primo-infection occurs during pregnancy. Furthermore it is a major cause of spontaneous abortions in sheep (estimated over 1 million lambs lost annually) and thus represents a serious veterinarian issue with huge economical consequences. T. gondii represents an appropriate model to study presentation of vacuolar antigens by MHC I molecules since CD8 T cells are known to be key for resistance. In addition, T. gondii is phylogenetically close to Plasmodium spp, the malaria-causing parasite responsible for 0.7 million deaths annually. As a genetically tractable microorganism, T. gondii is a model of choice to study intravacuolar apicomplexan parasites.In humans, the parasite antigens recognized by CD8 T cells during T. gondii infection are largely unknown. In mice, some of these antigens have been revealed but their processing pathways remain unclear. During our previous work, we have discovered the first natural CD8 T cell antigen from T. gondii in the mouse: a decameric peptide derived from the secretory protein GRA6 and presented by H2-Ld MHC I molecules. We showed that this immunodominant antigen induces a strong CD8 T cell response during infection and protects mice against lethal parasitic challenge. Since then, additional epitopes have been discovered by us and others.ObjectivesThe objectives of the APATRIVAP project were to elucidate the mechanisms of antigen processing and CD8 T cell recognition during infection by the T. gondii intravacuolar parasite. We have addressed 3 main questions:1/ What are the molecular bases of GRA6 epitope strong immunogenicity ?2/ What is the mechanism behind fusion of the parasitophorous vacuole and the host endoplasmic reticulum and what is its role on T. gondii antigen presentation ?3/ What is the impact of antigen intracellular transport on T. gondii antigen presentation ?Achievements1/ What are the molecular bases of GRA6 epitope strong immunogenicity ?We first ruled out two possible causes: peptide affinity for Ld and frequency of GRA6-specific T cells in the naive mouse. Using a variety of transgenic parasites, we established that presentation in vitro and in vivo is negatively affected when the epitope is no longer present at GRA6 C-terminus. Thanks to a newly generated T cell hybridoma against a subdominant T. gondii antigen, we reported that position of the subdominant epitope at GRA6 C-terminus enhanced immunogenicity and overturned immunodominance (Feliu et al, PLoS Pathogens 2013 9(6):e1003449). Our data provide crucial information on the molecular processes that make a protein from an intracellular parasite accessible for detection by CD8 T cells.2/ What is the mechanism behind fusion of the parasitophorous vacuole (PV) and the host endoplasmic reticulum (ER) and what is its role on T. gondii antigen presentation ?Similar to the ER-mediated phagocytosis phenomenon, fusion between PV and ER has been proposed to be involved in presentation of the parasite-derived OVA model antigen in dendritic cells (DC) (Goldzsmidt R et al, J Exp Med 2009). In collaboration with the lab of S. Amigorena at Institut Curie in Paris, we have examined the molecular basis of this process. Thanks to shRNA-mediated silencing of an ER-localized SNARE protein called Sec22b, we revealed the implication of Sec22b in recruitment of host endoplasmic reticulum (ER) components onto the PV membrane. By doing so, Sec22b participates in the control of MHC I presentation of OVA secreted by T. gondii in infected DC (Cebrian I et al, Cell 2011 147(6):1355-68).3/ What is the impact of antigen intracellular transport on T. gondii antigen presentation ?Within the vacuole, the GRA6 immunodominant antigen as well as some other antigenic proteins, are known to bind to a network of membrane tubules called Intravacuolar Tubulovesicular Network (IVN). Using reverse genetics to create parasite mutants with perturbed GRA6 localization, we have studied how parasite protein trafficking and membrane binding regulate antigen presentation. We first generated GRA2-deficient T. gondii mutants where IVN biogenesis is disrupted. With two independent T. gondii strains, we observed a strong increase in GRA6 MHC I presentation in the absence of IVN. We then approached the same question from a different angle, by perturbing intracellular transport of GRA6 directly (e.g. making GRA6 membrane-associated or soluble in the vacuole). We observed that membrane association is key for optimal presentation by infected cells. This work is currently prepared as a manuscript to be submitted for publication in Oct 2014 (Lopez et al, in preparation).SignificanceThis project synergized the fields of immunology, cell biology and parasitology. By uncovering novel parameters controlling immunogenicity of T. gondii parasite antigens, it will have implications for the design of a toxoplasmosis vaccine and/or for the amelioration of vaccines against other apicomplexan parasites. Additionally, our findings will be relevant for the optimization of T. gondii as a live vaccine vector inducing potent anti-tumor CD8 responses.
