Immunity to Toxoplasma gondii

AbstractImmunity to Toxoplasma gondiiby Harshita Satija GroverDoctor of Philosophy in Molecular and Cellular BiologyUniversity of California, BerkeleyProfessor Nilabh Shastri, Co-chairProfessor Ellen Robey, Co-chairToxoplasma gondii (T. gondii) is an obligate intracellular protozoan parasite, capable of infecting all warm-blooded animals, and can cause a severe disease in immunocompromised individuals. Protection to T. gondii is largely mediated by CD8 T cells, although CD4 T cells have also been shown to be important in immunity to T. gondii. However, the natural antigens recognized by these CD8 T cells, and how they mediate immune protection in vivo is poorly understood. Recently, it has been discovered that in mice with H-2d MHC molecules, there is an immunodominant CD8 T cell response to GRA6, a dense granule T. gondii protein. In the absence of the T cell response to GRA6, such as in ERAAP-/- animals, mice succumb to infection. On the contrary, C57BL/6 (H-2b) mice do not mount a response to GRA6. Studying antigen presentation dependent on MHC haplotype is important because the response to T. gondii in mice is controlled by genes in the H-2 region, wherein mice with the H-2b haplotype are more susceptible to infection than mice with H-2d MHC molecules. In addition, C57BL/6 (B6) mice are good models because a large number of genetic mutations in the innate and adaptive immune system have been established in this strain. To better understand T cell responses in the susceptible strain, we immunized B6 mice with T. gondii, and found that it leads to potent CD4 T cell but weak CD8 T cell response. To identify the CD4 T cell stimulating antigens, we generated a T. gondii-specific, lacZ inducible, CD4 T cell hybridoma and used it as a sensitive probe to screen a T. gondii-cDNA library. We isolated a cDNA encoding a putative secreted protein of unknown function that we named CD4Ag28m and identified the minimal peptide, AS15 that was presented by MHC class II molecules to the CD4 T cells. Immunization of mice with the AS15 peptide provided protection to subsequent parasite challenge, resulting in a lower parasite burden and cyst load.Furthermore, in order to characterize the CD8 T cell response to T. gondii in B6 mice, we restimulated T cells from mice immunized with T. gondii, with MHC Class II-/- bone marrow dendritic cells (BMDCs) to allow for the proliferation of CD8 T cells. This allowed us to generate a CD8 T cell hybridoma that was T. gondii specific and MHC Class I H-2Db restricted. This hybridoma was used as a probe to screen the T. gondii cDNA library, which revealed that it recognizes ROP5, a rhoptry protein from T. gondii. We identified that YAL9 is the minimal antigenic peptide recognized by CD8 T cells. In chronically infected mice, response towards YAL9 in the brain and spleen is minor but detectable, and unlike the protective GRA6 response in H-2d mice, immunization of B6 mice with YAL9 does not provide protection in B6 mice challenged with lethal dose of T. gondii. Most importantly, we found that altering the expression and trafficking of ROP5 from rhoptries to dense granules in parasites enhances the immunogenicity of ROP5 in B6 mice and now protects the mice from lethal challenge with transgenic parasites after peptide immunization.Identification of the antigens in the H-2b mice will enable us to study antigenic specific T cell responses, including effector functions such as clonal expansion and memory response at different stages of infection. We can now generate T cell receptor transgenic mice and use the antigen specific T cells from these mice to visualize and measure the dynamics of T cell interaction with antigen presenting cells during infection. Most significantly, understanding the nature of how these antigens are presented to T cells will allow us to design new and improved vaccine candidates against T. gondii and other apicomplexan parasites

Impact of Regulated Secretion on Antiparasitic CD8 T Cell Responses

CD8 T cells play a key role in defense against the intracellular parasite Toxoplasma, but why certain CD8 responses are more potent than others is not well understood. Here, we describe a parasite antigen, ROP5, that elicits a CD8 T cell response in genetically susceptible mice. ROP5 is secreted via parasite organelles termed rhoptries that are injected directly into host cells during invasion, whereas the protective, dense-granule antigen GRA6 is constitutively secreted into the parasitophorous vacuole. Transgenic parasites in which the ROP5 antigenic epitope was targeted for secretion through dense granules led to enhanced CD8 T cell responses, whereas targeting the GRA6 epitope to rhoptries led to reduced CD8 responses. CD8 T cell responses to the dense-granule-targeted ROP5 epitope resulted in reduced parasite load in the brain. These data suggest that the mode of secretion affects the efficacy of parasite-specific CD8 T cell responses

Dynamics of Neutrophil Migration in Lymph Nodes during Infection

Although the signals that control neutrophil migration from the blood to sites of infection have been well characterized, little is known about their migration patterns within lymph nodes or the strategies that neutrophils use to find their local sites of action. To address these questions, we used two-photon scanning-laser microscopy to examine neutrophil migration in intact lymph nodes during infection with an intracellular parasite, Toxoplasma gondii. We found that neutrophils formed both small, transient and large, persistent swarms via a coordinated migration pattern. We provided evidence that cooperative action of neutrophils and parasite egress from host cells could trigger swarm formation. Neutrophil swarm formation coincided in space and time with the removal of macrophages that line the subcapsular sinus of the lymph node. Our data provide insights into the cellular mechanisms underlying neutrophil swarming and suggest new roles for neutrophils in shaping immune responses