Abstract
Effective control of the intracellular protozoan parasite Toxoplasma gondii depends on the activation of antigen-specific CD8+ T-cells that manage acute disease and prevent recrudescence during chronic infection. T-cell activation in turn, requires presentation of parasite antigens by MHC-I molecules on the surface of antigen presenting cells. CD8+ T-cell epitopes have been defined for several T. gondii proteins, but it is unclear how these antigens enter into the presentation pathway. We have exploited the well-characterized model antigen ovalbumin (OVA) to investigate the ability of parasite proteins to enter the MHC-I presentation pathway, by engineering recombinant expression in various organelles. CD8+ T-cell activation was assayed using ‘B3Z’ reporter cells in vitro, or adoptively-transferred OVA-specific ‘OT-I’ CD8+ T-cells in vivo. As expected, OVA secreted into the parasitophorous vacuole strongly stimulated antigen-presenting cells. Lower levels of activation were observed using glycophosphatidyl inositol (GPI) anchored OVA associated with (or shed from) the parasite surface. Little CD8+ T-cell activation was detected using parasites expressing intracellular OVA in the cytosol, mitochondrion, or inner membrane complex (IMC). These results indicate that effective presentation of parasite proteins to CD8+ T-cells is a consequence of active protein secretion by T. gondii and escape from the parasitophorous vacuole, rather than degradation of phagocytosed parasites or parasite products.
Highlights
Class I Major Histocompatability Complex (MHC-I) molecules present peptides generated by proteasomal degradation in the cytosol and transport into the endoplasmic reticulum, or by crosspresentation of endo/phagocytosed material [1,2]
The route by which T. gondii antigens reach the endoplasmic reticulum for loading onto MHC-I is not fully understood, as these parasites reside within a specialized intracellular ‘parasitophorous vacuole’ (PV) distinct from the phagocytic/endocytic pathway and the host cell cytoplasm
In order to address the route of T. gondii antigen entry into the MHC-I presentation pathway, we have examined CD8+ T-cell activation following infection with parasites engineered to target the well-characterized antigen ovalbumin to various locations, including the parasite cytoplasm, mitochondrion, inner membrane complex, plasma membrane, and the parasitophorous vacuolar space
Summary
Class I Major Histocompatability Complex (MHC-I) molecules present peptides generated by proteasomal degradation in the cytosol and transport into the endoplasmic reticulum, or by crosspresentation of endo/phagocytosed material [1,2]. The route by which T. gondii antigens reach the endoplasmic reticulum for loading onto MHC-I is not fully understood, as these parasites reside within a specialized intracellular ‘parasitophorous vacuole’ (PV) distinct from the phagocytic/endocytic pathway and the host cell cytoplasm. In order to address the route of T. gondii antigen entry into the MHC-I presentation pathway, we have examined CD8+ T-cell activation following infection with parasites engineered to target the well-characterized antigen ovalbumin to various locations, including the parasite cytoplasm, mitochondrion, inner membrane complex, plasma membrane, and the parasitophorous vacuolar space
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