CD8⁺T Cells Specific for a Malaria Cytoplasmic Antigen Form Clusters around Infected Hepatocytes and Are Protective at the Liver Stage of Infection

Abstract

cFollowing Anopheles mosquito-mediated introduction into a human host, Plasmodium parasites infect hepatocytes and undergo intensive replication. Accumulating evidence indicates that CD8 ؉ T cells induced by immunization with attenuated Plasmodium sporozoites can confer sterile immunity at the liver stage of infection; however, the mechanisms underlying this protection are not clearly understood. To address this, we generated recombinant Plasmodium berghei ANKA expressing a fusion protein of an ovalbumin epitope and green fluorescent protein in the cytoplasm of the parasite. We have shown that the ovalbumin epitope is presented by infected liver cells in a manner dependent on a transporter associated with antigen processing and becomes a target of specific CD8؉ T cells from the T cell receptor transgenic mouse line OT-I, leading to protection at the liver stage of Plasmodium infection. We visualized the interaction between OT-I cells and infected hepatocytes by intravital imaging using two-photon microscopy. OT-I cells formed clusters around infected hepatocytes, leading to the elimination of the intrahepatic parasites and subsequent formation of large clusters of OT-I cells in the liver. Gamma interferon expressed in CD8 ؉ T cells was dispensable for this protective response. Additionally, we found that polyclonal ovalbumin-specific memory CD8 ؉ T cells induced by de novo immunization were able to confer sterile protection, although the threshold frequency of the protection was relatively high. These studies revealed a novel mechanism of specific CD8 ؉ T cell-mediated protective immunity and demonstrated that proteins expressed in the cytoplasm of Plasmodium parasites can become targets of specific CD8 ؉ T cells during liver-stage infection.

CD8+T Cells Eliminate Liver-Stage Plasmodium berghei Parasites without Detectable Bystander Effect

“…Nonetheless, a variety of lines of evidence support the notion that CD8 ϩ T cell killing occurs following cognate CD8 ϩ T cell-hepatocyte interactions. Recent independent in vivo imaging studies observed clustering of CD8 ϩ T cells around infected hepatocytes that led to the eventual killing of the parasites (10,11). Moreover, several in vitro and in vivo studies suggest that the infected cells themselves are able to present antigen directly to effector CD8 ϩ T cells (12)(13)(14)(15).…”

Local Immune Response to Injection of Plasmodium Sporozoites into the Skin

“…We used the P.berghei ANKA clone expressing GFP under the control of the hsp70 promoter (34), the P.berghei ANKA clone 676cl1 (Pb GFP-LUC sch ) expressing a GFP-luciferase fusion gene via the EEF1a promoter (35), the P. berghei ANKA clone deficient for SPECT2 (Sporozoite micronemal protein essential for cell traversal 2) and expressing GFP under the control of the hsp70 promoter (SPECT2F) (36), and the P.berghei ANKA clone PbA-hsOVA expressing class I and class II OVA epitopes (37).…”

TAP‐mediated processing of exoerythrocytic antigens is essential for protection induced with radiation‐attenuated Plasmodium sporozoites

“…In the event that Plasmodium infected hepatocytes are still intact, processing may involve a direct cytosolic pathway where Ags that cross the PV and enter the cytosol would be degraded by proteasomes and then be transported by TAP to the ER for loading onto the nascent MHC I molecules. Studies with bone marrow chimeras showed that hepatocytes infected with P. berghei sporozoites expressing recombinant cytoplasmic fusion protein OVA-Hsp70 needed the classical TAP-dependent MHC class I pathway to process and present the OVA epitope [25]. Conversely, it has been reported that normal, healthy hepatocytes have very low levels of cathepsin S [59], an essential enzyme for TAP-independent processing [10].…”

“…At present, however, the identity of LS Ags that induce protracted protection in RAS-immunized mice remains largely unknown. More importantly, apart from the processing and presentation of the circumsporozoite protein (CSP) [24], the major sporozoite surface Ag and recombinant cytoplasmic fusion protein OVA-Hsp70 [25], pathways of processing and presentation of EE Ags for the induction of CD8 + T cells have not been investigated in the malaria system. Like infectious sporozoites, RAS enter the liver and invade hepatocytes where they undergo arrested development into LS parasites that express LS-specific Ags, but do not infect red blood cells (RBC) and hence do not cause clinical malaria.…”