Subcellular Antigen Location Influences T-Cell Activation during Acute Infection with Toxoplasma gondii

et al. (2011) Subcellular Antigen Location Influences T-Cell Activation during Acute Infection with Toxoplasma gondii. PLoS ONE 6(7): e22936. doi:10.1371/journal.pone.0022936 Subcellular Antigen Location Influences T-Cell Activation during Acute Infection with Toxoplasma gondii Beth Gregg 0 Florence Dzierszinski 0 Elia Tait 0 Kimberly A. Jordan 0 Christopher A. Hunter 0 David S. 0 Yousef Abu Kwaik, University of Louisville, United States of America 0 1 Department of Biology, University of Pennsylvania, Philadelphia, Pennsylvania, United States of America, 2 Department of Pathobiology, University of Pennsylvania , Philadelphia, Pennsylvania , United States of America 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+ Tcell 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. - Funding: This work was supported by grants AI28724, AI071302, and AI 42334 from the National Institutes of Health (NIH). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. 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]. During infection, MHC-I antigen presentation is responsible for the activation and expansion of specific CD8+ T-cells, and is involved in the immune response to diverse intracellular pathogens, including viruses, bacteria, and microbial eukaryotes [3,4]. CD8+ T-cells are required for the control of the protozoan parasite Toxoplasma gondii during its acutely lytic tachyzoite stage, and (directly or indirectly) during the chronic bradyzoite stage characterized by latent cysts in the muscle, brain and other tissues [5,6]. Activated T-cells recognize and destroy both T. gondii parasites and parasite-infected cells, and also produce IFN-c, activating reactive oxygen pathways [79]. 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. Presentation is dependent on host cell immunoproteasomes, TAP, and ERAAP [1012], indicating that parasite antigens must reach the host cell cytosol, and several pathways have been proposed, including cross-presentation of phagocytosed parasite material, degradation of the PV membrane, secretion of parasite proteins outside of the PV, and fusion of the PV with the host cell ER [1315]. Various immunogenic T. gondii antigens are known, including proteins secreted from the dense granules and rhoptries, but responses are often both parasite and host strain-specific [9,12,16,17]. 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. Generation of transgenic parasite expressing organelle specific OVA antigen To explore whether antigen access to the MHC-I presentation pathway is affected by subcellular location of antigen within T. gondii, RH strain T. gondii was engineered to stably express the model antigen OVA (amino acids 140386), fused to various organelletargeting sequences as described under Methods. The rationale behind these experiments was to help distinguish between crosspresentation of phagocytosed antigen, versus translocation of antigen across the parasitophorous vacuole where intracellular T. gondii parasites reside (a compartment distinct from the endophagocytic system; [18]). As shown in Fig. 1A (top row), expression of ovalbumin without additional targeting signals results in cytoplasmic localization (Cyto-OVA), while fusion to a signal sequence results in secretion into the parasitophorous vacuole (P30-OVA), as previously described by Pepper et al [19]. Co-localization of additional OVA fusion proteins with well-characterized markers showed proper targeting to the inner membrane complex (IMCOVA, row 2), the mitochondrion (HSP-OVA, row 3), or the cell surface, using a GPI anchor (GPI-OVA, row 4). Antibodies to OVA label the surface of non-permeabilized, extracellular GPI-OVA parasites, indicating targeting to the parasite membrane (row 4); permeabilization prior to staining also reveals OVA associated with internal secretory organelles (ER, Golgi, vesicles), presumably en route to the plasma membrane (row 5). As antigen load is known to be important during infection [20], OVA levels were assessed by immunoblotting of parasites (Fig. 1B) and infected culture supernatants (Fig. 1C). Levels of OVA produced by individual parasite strains ranged from 4.5 to 21 ng/ 106 parasites. Secreted antigen was detected only in P30-OVA and GPI-OVA culture supernatants (Fig. 1C). We presume that significant quantities of GPI-OVA protein are shed from the parasite surface during parasite gliding motility, as has been reported for the endogenous P30 protein [21]. No secreted antigen was detected in culture supernatants following HSP-OVA or IMCOVA infection. Cyto-OVA was not included on this Western blot, but no secreted OVA was detected in Pru Cyto-OVA transgenics (not shown), and note that n (...truncated)