Plasmodium berghei-Infected Primary Hepatocytes Process and Present the Circumsporozoite to Specific CD8+ T Cells In Vitro This information is current as of September 23, 2021. Silayuv E. Bongfen, Ralph Torgler, Jackeline F. Romero, Laurent Renia and Giampietro Corradin J Immunol 2007; 178:7054-7063; ; doi: 10.4049/jimmunol.178.11.7054 http://www.jimmunol.org/content/178/11/7054 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2007 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Plasmodium berghei-Infected Primary Hepatocytes Process and Present the Circumsporozoite Protein to Specific CD8؉ T Cells In Vitro1

Silayuv E. Bongfen,* Ralph Torgler,* Jackeline F. Romero,* Laurent Renia,†‡§¶ and Giampietro Corradin2*

A substantial and protective response against liver stages is directed against the circumsporozoite protein (CSP) and involves induction of CD8؉ T cells and production of IFN-␥. CSP-derived peptides have been shown to be presented on the surface of infected hepatocytes in the context of MHC class I molecules. However, little is known about how the CSP and other sporozoite Ags are processed and presented to CD8؉ T cells. We investigated how primary hepatocytes from BALB/c mice process the CSP of Plasmodium berghei after live sporozoite infection and present CSP-derived peptides to specific Downloaded from H-2Kd-restricted CD8؉ T cells in vitro. Using both wild-type and spect؊/؊ P. berghei sporozoites, we show that both infected and traversed primary hepatocytes process and present the CSP. The processing and presentation pathway was found to involve the proteasome, Ag transport through a postendoplasmic reticulum compartment, and aspartic proteases. Thus, it can be hypothesized that infected hepatocytes can contribute in vivo to the elicitation and expansion of a T cell response. The Journal of Immunology, 2007, 178: 7054–7063. http://www.jimmunol.org/ he feasibility of vaccine development against malaria mice are not protected against sporozoite challenge, despite induc- liver stages is indicated by studies (1, 2) which show that tion of IL-12, IFN-␥, and proliferating T cells (15). sterile immunity against the liver stage can be obtained by It was widely believed that only endogenous Ags could be pre- T ϩ immunization of animals and humans with irradiated and, recently, sented on MHC class I molecules to CD8 T cells. A large body genetically attenuated sporozoites (3). Irradiated sporozoite-in- of evidence now shows that peptides derived from exogenous Ags duced protective immunity has been shown to involve IFN-␥, are also capable of binding to MHC class I molecules and stimu- CD4ϩ, and CD8ϩ T cells directed against liver stage parasites, and lating CD8ϩ T cells, a process known as cross-presentation. Sev- in vivo depletion of these effectors has led to blood infection upon eral models of cross-presentation have been described (16–21), a live sporozoite challenge (4–10). especially for professional APCs. In addition, the use of exogenous by guest on September 23, 2021 Even though sporozoites pass through Kupffer cells before in- Ags such as long synthetic peptides to immunize mice (22, 23) and fecting hepatocytes (11), hepatocytes are the only cells that sustain humans (24) to obtain MHC class I-restricted CTLs also supports complete development of malaria liver stages after infection with the concept of cross-presentation. Besides professional APCs, non- sporozoites. Maintenance of the anti-sporozoite protective CD8ϩ professional APCs have also been shown to be capable of cross- T cell response requires hepatic stages, since protection against presenting long polypeptides to CD8ϩ T cells (25, 26). sporozoite challenge is abrogated if these hepatic stages are elim- The mechanisms involved in malaria Ag presentation to T cells inated (12). Furthermore, immunization with irradiated Plasmo- remain unclear, because the low number of Plasmodium-infected dium berghei-infected hepatocytes (but not infected liver non- hepatocytes in the liver has prevented the development of direct parenchymal cells) conferred protection against a sporozoite Ag presentation studies. The question is further complicated by the challenge (13). It has been suggested that in malaria, hepatocytes fact that sporozoites do not infect the first hepatocyte they encoun- express MHC class I-peptide complexes on their surface and that ter, but traverse through many hepatocytes before infecting a final the recognition of these complexes by CD8ϩ T cells is necessary one (27). As they traverse, they leave behind trails of the circum- for protection (reviewed in Ref. 14). This notion is supported by sporozoite protein (CSP)3 in the traversed cells, some of which studies which show that irradiated sporozoite-immunized ␤2mϪ/Ϫ reseal their membranes and survive and are, in principle, capable of presenting the Ag. This suggests that sporozoite Ags could ei- ther be presented by the infected cell, the traversed cell, or both. It has been suggested (28) that migration of sporozoites through *Department of Biochemistry, University of Lausanne, Epalinges, Switzerland; †In- hepatocytes is essential for final infection. However, a recent stitut Cochin, Departement d’Immunologie, Hoˆpital Cochin, Paris, France; ‡Institut National de la Sante´et de la Recherche Me´dicale, Paris, France; §Centre National de study (29) has identified a sporozoite microneme protein essen- la Recherche Scientifique Unite´Mixte de Recherche, Paris, France; and ¶Universite´ tial for cell traversal (SPECT) and has shown that sporozoites Rene Descartes, Paris, France Received for publication June 28, 2006. Accepted for publication March 19, 2007. The costs of publication of this article were defrayed in part by the payment of page 3 Abbreviations used in this paper: CSP, circumsporozoite; SPECT, sporozoite mi- charges. This article must therefore be hereby marked advertisement in accordance croneme protein essential for cell traversal; wt, wild type; BFA, brefeldin A; LLnL, with 18 U.S.C. Section 1734 solely to indicate this fact. N-acetyl-L-leucyl-L-leucyl-L-norleucinal; Cbz-LLL, carbobenzoxyl-leucinyl-leucinyl- Ј Ј 1 leucinal; ICS, intracellular cytokine staining; RT, room temperature; DAPI, 4 ,6 - This work was supported by European Community Grant QLK 2-CT-2002-00700. diamidino-2-phenylindole; EEF, exoerythrocytic form; GdCl, gadolinium chloride; 2 Address correspondence and reprint requests to Dr. Giampietro Corradin, Depart- DC, dendritic cell; HSP, heat shock protein. ment of Biochemistry, Chemin des Boveresses 155, Epalinges, Switzerland. E-mail address: [email protected] Copyright © 2007 by The American Association of Immunologists, Inc. 0022-1767/07/$2.00 www.jimmunol.org The Journal of Immunology 7055 deficient in the spect gene are incapable of cell traversal, but capable of infection and normal development. spectϪ/Ϫ para- sites are therefore an essential tool in determining the ability of the infected hepatocyte to process and present sporozoite Ags to CD8ϩ T cells. Despite evidence for the importance of hepatic stages to maintain a protective immune response and suggestions that hepatocytes present MHC class I-peptide complexes on their surface, very few studies (30, 31) have attempted to address the question of direct Ag presentation by hepatocytes in an in vitro or in vivo assay. In this study, using mouse primary hepatocytes, wild-type (wt) and spectϪ/Ϫ P. berghei sporozoites, we investigated the cell (tra- versed or infected hepatocytes or both) involved in processing and presentation of the CSP of P. berghei to specific CD8ϩ T cells. We also investigated the pathway of this processing and presentation by the primary hepatocytes after infection with wt sporozoites. We FIGURE 1. Primary hepatocytes optimally process and present CSP- formally show that both infected and traversed hepatocytes are able to derived peptides 8–20 h after infection with sporozoites. Primary hepato- process the native CSP present on live parasites for presentation to cytes were infected with P. berghei sporozoites for different lengths of ϩ specific CD8 T cells. Our data show that this processing is predom- time. Secretion of IFN-␥ by specific CD8ϩ T cells after coculture with Downloaded from inantly dependent on the proteasome and also on aspartic proteases. infected hepatocytes was determined by an IFN-␥ ELISPOT assay. Error bars, Mean Ϯ SD of duplicate wells. Materials and Methods Peptides Ϫ/Ϫ d Anopheles stephensi mosquitoes containing either wt or spect (29) P. The peptide PbCS245–253, representing the H-2K -restricted CTL epitope of berghei sporozoites were maintained under the same conditions. Sporozo- http://www.jimmunol.org/ the CSP of P. berghei and the peptide PfCS327–335, an HLA-A2.1-restricted Ϫ/Ϫ peptide from the CSP of P. falciparum, were synthesized by standard solid- ites (wt and spect ) were obtained by dissection of infected female A. phase F-moc chemistry. Peptide stock solutions (2 mg/ml) were prepared stephensi mosquitoes to obtain salivary glands, followed by homogeniza- in 1ϫ PBS and stored at Ϫ20°C. tion of the salivary glands to release sporozoites. Wild-type midgut sporo- zoites were also used. Cells Inhibitors The APCs were primary hepatocytes isolated from BALB/c mice by a The inhibitors used were brefeldin A (BFA), N-acetyl-L-leucyl-L-leucyl-L- two-step perfusion of liver lobules with collagenase as previously de- norleucinal (LLnL), carbobenzoxyl-leucinyl-leucinyl-leucinal (Cbz-LLL), scribed (32), with slight modifications. Briefly, mice were sacrificed by lactacystin, pepstatin, leupeptin, chloroquine, and ammonium chloride CO2 inhalation, dissected, and a liver lobule was cut out. The lobule was ϩ (NH Cl). All inhibitors were obtained from Sigma-Aldrich. In preliminary by guest on September 23, 2021 perfused for 10 min with Ca2 -free HEPES buffer (pH 7.6) at 37°C and at 4 experiments, we showed that none of these inhibitors were toxic for the a rate of 5 ml/min. The lobule was then perfused with type IV collagenase ϩ primary hepatocytes at the concentrations used (data not shown). (Sigma-Aldrich) solution (Ca2 -free HEPES buffer containing 0.04% type ⅐ IV collagenase and 0.075% CaCl2 2H2O) for 5 min at 37°C. The perfused IFN-␥ ELISPOT assay lobule was incubated for 10 min at 37°C in the collagenase solution. Using sterile pipettes, the tissue was gently teased apart to release cells. Cells The IFN-␥ ELISPOT assay was performed as previously described (33). ϩ were washed once with Ca2 -free HEPES buffer at 800 rpm for 30 s at Briefly, 105 primary hepatocytes were seeded per well in 1% gelatin-coated 4°C. The pellet was gently resuspended in DMEM, layered on 60% Percoll, 48-well culture plates and incubated overnight at 37°C to enable them to and centrifuged at 2000 rpm for 2 min at 4°C. The resulting pellet was adhere. Hepatocytes were washed with culture medium and infected for resuspended in complete culture medium (DMEM supplemented with 10% different lengths of time with P. berghei sporozoites (25,000/well). Unin- FCS, 1% penicillin-streptomycin, 1% HEPES, and 0.05 mM 2-ME), and fected cells were used as a negative control. All cells were washed twice centrifuged again at 800 rpm for 30 s at 4°C. The pellet was resuspended with culture medium and cocultured with 5000 PbCS-C7 CD8ϩ T cells per in complete culture medium, 105 cells seeded per well in a 48-well gelatin- well for4hat37°C. T cells were then transferred to ELISPOT plates coated culture plate and incubated at 37°C. Two to 4 h later, the medium previously coated with anti-mouse IFN-␥ Ab and blocked with 1% BSA. was replaced by fresh culture medium and the cells were incubated over- The ELISPOT plates were incubated for 20–24 h at 37°C, after which the night at 37°C. plates were washed and developed. Spots were visualized and counted with To ensure the purity of the hepatocyte preparation, the isolated hepato- an automated ELISPOT reader. cytes were stained with a panel of Abs to exclude the presence of contam- inating cells (anti-F4/80 Ab conjugated to FITC to exclude macrophages, Intracellular cytokine staining (ICS) anti-CD11c Ab conjugated to PE to exclude dendritic cells (DCs), and 5 anti-MHC class II Ab conjugated to PE to exclude both macrophages and Primary hepatocytes (10 ) were seeded per well in 1% gelatin-coated 48- well culture plates and incubated overnight at 37°C. Cells were washed and DCs) and also positively stained with anti-rabbit ferritin Ab, using rabbit Ϫ/Ϫ anti-mouse IgG Alexa Ab conjugated to Alexa Fluor 488 as a second Ab. infected with 25.000 wt, spect , or midgut sporozoites per well for 8 h As a positive control for the anti-F4/80 Ab, peritoneal macrophages were at 37°C. As controls, heat-killed wt sporozoites or material from salivary used, whereas DCs were used as a positive control for the anti-CD11c and glands of uninfected mosquitoes were incubated with hepatocytes for 8 h before coculture with specific CD8ϩ T cells. As a positive control, cells anti-MHC class II Abs. The cells were then analyzed by FACS after stain- ␮ ing (data not shown). were pulsed with 1 g/ml of the PbCS245–253 peptide for2hat37°C. Unpulsed/uninfected cells were used as a negative control. After pulsing The human hepatoma cell line HepG2 was used in certain experiments. ϩ d ϩ P. and infection, cells were washed and cocultured with 5000 PbCS-C7 CD8 The H-2K -restricted CD8 T cell clone PbCS-C7, specific for the ␮ berghei CSP CTL epitope PbCS (5), was used as effector cells. T cells per well for4hat37°C in the presence of 10 g/ml of the intra- 245–253 cellular transport inhibitor BFA. T cells were then harvested, washed, and Mice and sporozoites stained with FITC-conjugated anti-mouse CD8 Ab (BD Biosciences) for 30 min at 4°C. Cells were washed twice with PBS-3% FCS and fixed for Six- to 8-wk-old BALB/c and C57BL/6 mice were obtained from Harlan 10 min at room temperature with prewarmed 4% paraformaldehyde. After Breeders. All mice were housed under pathogen-free conditions in the an- two washes, with 0.5% saponin-1ϫ PBS, cells were stained overnight at imal facility of the Swiss Institute for Experimental Cancer Research (Ep- 4°C with PE-conjugated anti-mouse IFN-␥ Ab (BD Biosciences) diluted in alinges, Switzerland). Animals were handled according to institutional the saponin solution. Cells were washed with PBS-3% FCS and analyzed guidelines. for CD8ϩ and IFN-␥ϩ T cells by flow cytometry. 7056 PRESENTATION OF SPOROZOITE Ags BY HEPATOCYTES Downloaded from http://www.jimmunol.org/ by guest on September 23, 2021

FIGURE 2. Primary hepatocytes specifically process and present the CSP of P. berghei. H-2Kd hepatocytes were prepared from wt mice and from mice 5 treated with 10 mg/kg body weight of GdCl. Hepatocytes (10 /well) were either infected with P. berghei sporozoites (a) or pulsed with the PbCS245–253 peptide (b), then cocultured for 4 h with 5 ϫ 103 CD8ϩ T cells. c, H-2Kd hepatocytes (105) were either infected with sporozoites or incubated with material from uninfected mosquito salivary glands (sgm) before coculture with 5 ϫ 103 CD8ϩ T cells. d, Hepatocytes were either infected with P. berghei sporozoites or incubated with heat-inactivated (HK spz) or midgut sporozoites, then cocultured with CD8ϩ T cells. e, Hepatocytes were prepared from either BALB/c (H-2Kd) or C57BL/6 (H-2Kb) mice. They were infected with P. berghei sporozoites and cocultured with CD8ϩ T cells. Activation of the T cells was determined by ICS and flow cytometry. Error bars, Mean Ϯ SD of duplicate wells. Negative, Uninfected and unpulsed cells.

In separate experiments, primary hepatocytes were prepared from a ites. Pulsing or infection of inhibitor-treated cells was performed either BALB/c mouse 24 h after i.v. injection of 10 mg/kg body weight of gad- without washing off the inhibitor or after washing. Cells were then washed olinium chloride (GdCl) to depress Kupffer cell function (34). These hepa- and cocultured with specific CD8ϩ T cells. The inhibition assays were tocytes were then plated on 1% gelatin-coated 48-well plates and ICS was completed as described for ICS. conducted as described above. Chromium release assay Inhibition assay Primary hepatocytes (105) were plated per well in a 1% gelatin-coated In the inhibition assays, the inhibitors were used at the following concen- 96-well plate and incubated overnight at 37°C. They were washed and ␮ ␮ ␮ ␮ trations: LLnL (50 M), Cbz-LLL (10 M), lactacystin (20 M), pepstatin either pulsed with 1 g/ml of the PbCS245–253 peptide (using the HLA- ␮ ␮ ␮ ␮ (36 M), leupeptin (110 M), BFA (35 M), chloroquine (10 M), and A2.1-restricted CTL epitope from the CSP of P. falciparum, PfCS327–335, NH4Cl (10 mM). Hepatocytes were incubated for2hat37°C with the as a negative control) for1hat37°C, or infected with 25,000 wt sporo- different inhibitors before pulsing with peptide or infection with sporozo- zoites for 16 h. Seventy-five microcuries of chromium was then added and The Journal of Immunology 7057 Downloaded from http://www.jimmunol.org/ by guest on September 23, 2021 FIGURE 3. Both infected and uninfected (traversed) hepatocytes process and present the CSP. a, HepG2 cells were treated with chromium and infected with either wt or spectϪ/Ϫ sporozoites. Migration of sporozoites through cells was determined by quantifying the amount of chromium released. b, Primary hepatocytes were infected with either wt or spectϪ/Ϫ sporozoites. The number of EEFs was determined by staining intracellular parasites with an anti-HSP70 Ab. c, Primary hepatocytes were infected with either wt or spectϪ/Ϫ sporozoites. After coculture with specific CD8ϩ T cells, activation of T cells was determined by ICS and flow cytometry. Error bars, Mean Ϯ SD of duplicate wells. Negative, Uninfected cells. d, Primary hepatocytes were infected with either wt or spectϪ/Ϫ sporozoites, then cocultured with T cells at different E:T ratios for 20 h. The number of EEFs was determined for each condition. Error bars, Mean Ϯ SD of duplicate wells. incubation was continued for another hour. Cells were washed and cocul- tection of both extracellular and intracellular sporozoites) or with cold 1% tured with PbCS-C7 CTLs at different E:T ratios in the presence or absence glutaraldehyde (for detection of extracellular sporozoites only) for 10 min. of 10 ␮g/ml Fas-Fc for4hat37°C. The supernatants were collected and All cells were washed and blocked for1hatRTwith 1% BSA-1ϫ PBS, read with a gamma counter. followed by incubation with an anti-PbCS repeat Ab for1hatRT.After washing, cells were stained for1hatRTwith a 1/100 dilution of anti-mouse Detection of apoptotic hepatocytes IgG Ab conjugated to Alexa Fluor 488. Cells were then washed, mounted in ϫ 4 50% glycerol, and observed under a fluorescence microscope. Sporozoites Primary hepatocytes (8 10 ) were seeded per well in 8-well LabTek were counted in duplicate wells. The number of extracellular sporozoites (glu- chamber slides and incubated overnight at 37°C. Cells were washed and taraldehyde fixation) was subtracted from the number of sporozoites in wells ϫ 4 P. berghei either infected with 2 10 sporozoites for8horpulsed with with methanol fixation to yield the number of intracellular sporozoites. the PbCS245–253 peptide for 2 h at 37°C. Hepatocytes were washed and cocultured with PbCS-C7 CTLs at a ratio of 1:1 for2hat37°C. The CTLs Quantification of exoerythrocytic forms (EEFs) were washed off with 1ϫ PBS and the hepatocytes were fixed for 10 min with ice-cold methanol. After washing, cells were blocked with 1% Primary hepatocytes (8 ϫ 104) were seeded per well in 8-well LabTek BSA-1ϫ PBSfor1hatroom temperature (RT). Hepatocytes infected with chamber slides and incubated overnight at 37°C. Cells were infected with sporozoites were stained for the intracellular parasite by incubation with 2 ϫ 104 wt or spectϪ/Ϫ P. berghei sporozoites per well for 20 h at 37°C. anti-heat shock protein (HSP) 70 Ab (2E6) (35) for1hatRT,followed by Cells were washed with 1ϫ PBS and fixed for 10 min with ice-cold meth- washing and staining for1hatRTwith a 1/100 dilution of anti-mouse IgG Ab anol. Cells were blocked with 1% BSA-1ϫ PBSfor1hatRTfollowing conjugated to Alexa Fluor 488. Cells were washed and all cells were stained washing with 1ϫ PBS. Infected hepatocytes were then stained with anti- with a 1/100 dilution of 4Ј,6Ј-diamidino-2-phenylindole (DAPI) to detect ap- HSP70Abfor1hatRT.After washing, cells were stained for1hatRT optotic nuclei. The cells were then viewed under a fluorescence microscope. with a 1/100 dilution of anti-mouse IgG Ab conjugated to Alexa Fluor 488. Cells were then washed, mounted in 50% glycerol, and observed under a Quantification of intracellular sporozoites fluorescence microscope. EEFs were counted over the entire well.

4 Primary hepatocytes (8 ϫ 10 ) were seeded per well in 8-well LabTek Quantification of the migration capacity of sporozoites chamber slides and incubated overnight at 37°C. Cells were pretreated with different inhibitors for2hat37°C. Wild-type sporozoites (2 ϫ 104) were Sporozoites migrate through several hepatocytes breaching their plasma added without washing off inhibitors and incubated for2hat37°C. Cells membranes, before infecting a final one by formation of a parasitopho- were washed with 1ϫ PBS and either fixed with cold methanol (for de- rous vacuole (27). The wounded cells release cytosolic contents into the 7058 PRESENTATION OF SPOROZOITE Ags BY HEPATOCYTES microenvironment (R. Torgler, S. E. Bongfen, J. F. Romero, A. Tardivel, M. Thome, and G. Corradin, submitted for publication). We exploited this ability of sporozoites to wound cells and release cytosolic contents to quan- tify their migration capacity. HepG2 cells (105) were seeded per well in a 96-well plate and incubated for 48 h at 37°C to enable them to adhere. Seventy-five micorcuries of chromium was then added and incubated for1hat37°C. Cells were washed several times to eliminate all residual chromium, then infected with 5 ϫ 104 wt or spectϪ/Ϫ sporozoites for3hat37°C. The resulting super- natant was collected and read with a gamma counter. The amount of chro- mium released was proportional to the number of sporozoites added and hence to hepatocytes wounded due to the migratory action of sporozoites.

Statistical analysis The one-way ANOVA and two-tailed Student t test were used to determine the significance of variation between test samples. All p values Յ0.05 were considered to be significant.

Results Primary hepatocytes can process and present the P. berghei

CSP, and do so optimally 8–20 h after infection with Downloaded from sporozoites Our preliminary in vitro data showed that primary hepatocytes are capable of processing and presenting sporozoite Ags after infec- tion (data not shown). However, the level of CD8ϩ T cell activa- tion varied with the length of time of hepatocyte infection. To find

out the optimal time necessary for processing and presentation of http://www.jimmunol.org/ the CSP after infection with sporozoites, hepatocytes were infected with sporozoites for different time periods over a total period of 24 h. Secretion of IFN-␥ by the PbCS-C7 CD8ϩ T cells after coculture with infected hepatocytes was determined by an ELISPOT assay. Optimal activation of the T cells occurred when hepatocytes were infected with the sporozoites for a time period between 8 and 20 h. Acti- vation was lower when T cells were cocultured with hepatocytes infected with sporozoites for Ͻ8horϾ20 h. (Fig. 1). This sug-

FIGURE 4. Proteasome inhibitors and BFA inhibit processing and pre- by guest on September 23, 2021 gests that the optimal time necessary for hepatocytes to process ϩ sentation of the CSP. Primary hepatocytes were treated with the different and present the CSP to CD8 T cells after infection with sporo- inhibitors 2 h before infection with sporozoites (spz; a) or pulsing with the zoites is between 8 and 20 h. PbCS245–253 peptide (b). Infected and pulsed cells were cocultured with To establish the purity of the hepatocyte preparation, primary 5 ϫ 103 CD8ϩ T cells for4hat37°C. Activation of CD8ϩ T cells was hepatocytes were stained with different Abs (see Materials and determined by ICS and flow cytometry. Negative, Untreated cells. Error Methods) to exclude the presence of contaminating cells, and the bars, Mean Ϯ SD of duplicate wells. preparation was found to contain essentially hepatocytes (data not shown). To further prove that the Ag presentation was performed by hepatocytes and not by Kupffer cells that could contaminate the mice (H-2Kb). As can be seen in Fig. 2e, T cells were only acti- hepatocyte preparation, primary hepatocytes were prepared from a vated when they were cocultured with hepatocytes from BALB/c BALB/c mouse 24 h after i.v. injection of 10 mg/kg body weight mice, which express the correct MHC molecules (H-2Kd). of GdCl. GdCl is known to deplete Kupffer cells in vivo (34). Hepatocytes from both a wt and GdCl-treated mouse were then Both infected and wounded (traversed) hepatocytes process and used in ICS. As shown in Fig. 2a, hepatocytes depleted of Kupffer present the CSP cells presented the CSP of P. berghei just as well as hepatocytes Sporozoites migrate through several hepatocytes before infecting a from wt mice. Similarly, presentation of the PbCS245–253 peptide final one by formation of a parasitophorous vacuole (27). During by both preparations of hepatocytes was comparable (Fig. 2b). migration, they leave behind a trail of the CSP. Some of the tra- Taking into consideration that T cells could be activated by versed cells reseal their membrane and survive, and thus can, in contaminants of parasite preparations, hepatocytes were incubated principle, present the CSP to T cells. with material from uninfected mosquito salivary glands as a con- To find out which cells were presenting the CSP (traversed or trol. Fig. 2c shows that the activation of T cells observed is specific infected cells or both), an ICS assay was done with both wt and for sporozoites, because T cells were not activated in the presence spectϪ/Ϫ sporozoites, whose migratory ability through hepatocytes of uninfected salivary gland material. To eliminate the possibility is totally or profoundly inhibited (Fig. 3a) (29), but which infect of Ag from extracellular degradation being presented, hepatocytes and develop normally in hepatocytes (Fig. 3b). As shown in Fig. were incubated with heat-killed and with midgut sporozoites 3c, T cells are activated when hepatocytes are infected with either (which contain the mature CSP, but neither migrate through nor wt or spectϪ/Ϫ sporozoites. The level of activation of T cells by wt infect hepatocytes). T cells were not activated in either case, implying sporozoites is about 5-fold that observed with spectϪ/Ϫ sporozo- that migration and/or infection of sporozoites are necessary for the ites. Given that spectϪ/Ϫ parasites are capable of infection but not CSP to be processed and presented by hepatocytes (Fig. 2d). migration, and that migration and/or infection are necessary for To further demonstrate that hepatocytes are indeed presenting CSP processing and presentation (Fig. 2d), these results suggest the peptide, ICS was performed with hepatocytes from C57BL/6 that both infected and uninfected (traversed) hepatocytes present The Journal of Immunology 7059

and cocultured with specific CD8ϩ T cells at different E:T ratios for 20 h. The number of EEFs in each case was then determined. Fig. 3di shows that EEFs from hepatocytes infected with wt and spectϪ/Ϫ sporozoites are equally eliminated, indicating that in- fected cells do present the CSP. The perfect correlation (r ϭ 0.997) between the elimination of EEFs from wt or spectϪ/Ϫ sporozoite- infected cells as a function of E:T ratio (Fig. 3dii) indicates a null or limited contribution of parasite killing due to activation of CD8ϩ T cells by traversed cells.

Processing and presentation of the CSP by primary hepatocytes are dependent on the proteasome and on vesicular transport through the Golgi apparatus The presentation of proteasome-generated cytosolic peptides by MHC class I molecules requires vesicular transport from the en- doplasmic reticulum through the Golgi complex to the cell surface. The dependence of the processing of the CSP on the proteasome was tested using the proteasome inhibitors LLnL, Cbz-LLL, and Downloaded from lactacystin. Processing and presentation of the CSP after infection of hepatocytes with sporozoites was strongly inhibited ( p Ͻ 0.05) by the proteasome inhibitors (Fig. 4a). In contrast, presentation of

the PbCS245–253 peptide was unaffected (Fig. 4b). Likewise, sporo- zoite-infected hepatocytes lost their capacity to present the CSP when they were treated with the intracellular transport inhibitor BFA, http://www.jimmunol.org/ whereas presentation of the PbCS245–253 peptide was not affected. These results indicate that after sporozoite infection, processing and presentation of the CSP follow a proteasome-dependent pathway. To establish that the inhibitors added did not affect the biosyn- thetic machinery of the parasite, inhibitor-treated cells were washed before infection, and ICS was conducted as before. No difference in activation of T cells was observed, whether or not the inhibitors were washed before infection (data not shown), indicat-

ing that the inhibitors do not have an effect on the parasite, but by guest on September 23, 2021 FIGURE 5. Effect of lysosomal protease inhibitors on processing of the CSP. Primary hepatocytes were treated with the different inhibitors 2 h inhibit a process in the hepatocyte. before infection with P. berghei sporozoites (a) or pulsing with the

PbCS245–253 peptide (b). After coculture of infected and peptide-pulsed Effect of lysosomal protease inhibitors and inhibitors of hepatocytes with 5 ϫ 103 PbCS-C7 CD8ϩ T cells for 4 h, T cell activation lysosomal acidification on processing of the CSP was determined by ICS and FACS analysis. Negative control, Unpulsed and uninfected cells. Error bars, Mean Ϯ SD of duplicate wells. Sporozoites are a source of exogenous Ags and, as shown in other systems (26), it is possible that processing of sporozoite the CSP to T cells, with most of the presentation being done by the could depend, at least in part, on the vacuolar pathway. To test the traversed cells. To further show that infected cells present the CSP, involvement of endolysosomal proteases in CSP processing, hepa- hepatocytes were infected with either wt or spectϪ/Ϫ sporozoites tocytes were treated with leupeptin and pepstatin before infection

FIGURE 6. Inhibitors do not affect sporozoite infectiv- ity. Primary hepatocytes were infected with P. berghei sporozoites in the presence or absence of inhibitors. Num- ber of intracellular sporozoite was determined by staining parasites with an anti-P. berghei CSP repeat Ab. 7060 PRESENTATION OF SPOROZOITE Ags BY HEPATOCYTES Downloaded from http://www.jimmunol.org/ by guest on September 23, 2021

FIGURE 7. Lysis of hepatocytes by CD8ϩ T cells. a, CD8ϩ T cells kill only target cells pulsed with the specific peptide in a chromium release assay. Ϯ Error bars, Mean SD of duplicate wells. b, Primary hepatocytes were pulsed with the specific PbCS245–253 peptide and cocultured with PbCS-C7 CTLs at a ratio of 1:1. Apoptotic nuclei were detected by DAPI staining. c, Hepatocytes were infected with sporozoites in the presence or absence of specific peptide. They were cocultured with CD8ϩ T cells at different E:T ratios. Specific lysis was determined in a chromium release assay. Error bars, Mean Ϯ SD of duplicate wells. d, Primary hepatocytes were infected with sporozoites in the presence or absence of the specific peptide, then cocultured with specific CTLs (PbCS-C7) at a ratio of 1:1 for 2 h. Apoptotic nuclei were detected by DAPI staining (blue), whereas the intracellular parasite was detected with anti-HSP70 Ab (green). White arrowheads, Apoptotic nuclei.

with sporozoites or pulsing with the PbCS245–253 peptide. Process- tation of the PbCS245–253 peptide was not affected by any of the ing and presentation of the CSP were not significantly affected by inhibitors (Fig. 5b). leupeptin ( p Ͼ 0.05). However, activation of T cells was signifi- cantly reduced ( p Ͻ 0.05) when hepatocytes were treated with the The different inhibitors used have no effect on sporozoite aspartic protease inhibitor pepstatin. To further establish the role of infection of hepatocytes the endosomal compartment, the hepatocytes were treated with To show that the inhibitors which had an effect on processing and chloroquine and ammonium chloride, which alter the acidification presentation did not affect the rate of hepatocyte infection by of lysosomes and endolysosomes. No significant effect on process- sporozoites, hepatocytes were infected with sporozoites in the ing and presentation was observed (Fig. 5a). As expected, presen- presence or absence of inhibitors for 2 h. Infected cells were The Journal of Immunology 7061 stained for intracellular sporozoites using a mAb against the re- the crucial, still unresolved question is: can infected hepatocytes peats of the P. berghei CSP. As shown in Fig. 6, the number of present Ags to T cells in a primary and secondary response? Our intracellular sporozoites did not vary significantly ( p Ͼ 0.05) data formally prove that at least in vitro, both infected and tra- whether or not infection was done in the presence of inhibitors. versed hepatocytes can present sporozoite-derived Ags to cloned ϩ Ϫ/Ϫ ϩ CD8 T cells. spect P. berghei sporozoites have been shown to Hepatocytes are lysed by CD8 T cells after pulsing with the be incapable of cell traversal, but capable of infection and normal PbCS CTL epitope or after incubation with sporozoites development in hepatocytes (29). Our finding that CD8ϩ T cells In addition to production of cytokines such as IFN-␥, CD8ϩ T could be activated when hepatocytes were infected with spectϪ/Ϫ cells also induce target cell death via a perforin/granzyme B- or sporozoites (Fig. 3c) shows that infected hepatocytes present the P. Fas-dependent mechanism. To further prove that hepatocytes are berghei CSP CTL epitope to T cells, because in this case we have able to process and present the CSP after infection and to show that a situation of infection only and no or minimal traversal. This is the CD8ϩ T cells used were functional, chromium release assays further proven by the fact that in the presence of T cells, EEFs were conducted to find out whether hepatocytes could be lysed by from spectϪ/Ϫ parasites could be eliminated as well as those from CD8ϩ T cells after pulsing with the optimal CTL epitope or after wt parasites (Fig. 3d). The activation observed is unlikely to be due infection with sporozoites. to uptake and processing of extracellular CSP by hepatocytes,

Hepatocytes pulsed with the specific PbCS245–253 peptide were because no activation of T cells was observed when hepatocytes found to be efficiently lysed by specific CD8ϩ T cells, as opposed were incubated with heat-killed or midgut sporozoites (Fig. 2d). to hepatocytes pulsed with a CTL epitope of P. falciparum, The long-lasting protection observed with the irradiated sporozoite Downloaded from PfCS327–335, even at high E:T ratios (Fig. 7a). The killing of pep- model is also consistent with continuous presentation of sporozoite- tide-pulsed hepatocytes observed here seemed to be primarily due derived Ags by infected hepatocytes to primed T cells. It has been, to perforin/granzyme B, and not via Fas-Fas ligand, because ad- in fact, shown that as early as 12 h after infection, no liver mono- dition of soluble Fas-Fc did not inhibit killing. This killing was nuclear cells contain parasite Ags (38), suggesting that the only further shown by staining the peptide-pulsed hepatocytes for ap- cells that contain parasite Ags for a long period of time are hepa- optotic nuclei 2 h after coculture with specific CTLs. In this case, tocytes. In addition, elimination of hepatic stages after irradiated massive apoptosis was observed, as shown by the presence of frag- sporozoite immunization abrogates protection (12). More recently, http://www.jimmunol.org/ mented nuclei (Fig. 7b, fourth panel). No apoptosis was observed long-lasting protection has been obtained by immunization with when hepatocytes were cocultured with CTLs in the absence of live sporozoites of mice under chloroquine treatment (39). This peptide (Fig. 7b, third panel) or when peptide alone was added to protection was abrogated by eliminating hepatic forms with pri- hepatocytes (Fig. 7b, second panel). maquine. Although the role of primaquine in Ag presentation in Hepatocytes were also lysed by specific CTLs after infection vivo has not been elucidated, these data suggest that activation of with sporozoites, as shown by a chromium release assay (Fig. 7c). primary responses may be mediated by infected hepatocytes. It is The lysis was greatly increased when the specific peptide was known that priming of immune responses requires expression of added to infected hepatocytes. However, by microscopically ex- costimulatory molecules by the APCs. In fact, costimulatory mol- amining cocultures of CD8ϩ T cells and hepatocytes after sporo- ecules have been shown to be expressed in the cytoplasm of hepa- by guest on September 23, 2021 zoite infection, we found that infected hepatocytes did not appear tocytes during hepatitis C viral infection (40) and in biliary atresia to undergo apoptosis, as shown by the absence of apoptotic nuclei patients with liver dysfunction (41). It is, therefore, possible that in infected cells 2 h after coculture with specific CTLs in the pres- sporozoite infection of hepatocytes induces expression of costimu- ence (Fig. 7d, fourth panel) or absence (Fig. 7d, third panel)of latory molecules necessary for priming T cell responses. peptide. Interestingly, the uninfected cells bordering infected cells Although not formally proven, our in vitro microscopy data were apoptotic whether or not peptide was added (Fig. 7d, third show that sporozoite-infected hepatocytes do not undergo nuclear and fourth panels, arrowheads). Importantly, no apoptosis was ob- fragmentation, suggesting that they are resistant to CTL-induced served when infected cells were cultured alone (Fig. 7d, first apoptosis. This is in conformity with studies which have shown panel) or in the presence of IFN-␥ for 2 h (Fig. 7d, second panel). that infected cells are protected from apoptosis (42, 43). In addi- tion, the finding that Fas- and perforin-deficient mice could still be Discussion protected from P. berghei (44) and P. yoelii (45) infection suggests The aim of this study was to provide evidence that primary hepa- that other mechanisms are implicated in the destruction of hepatic tocytes are able to process the P. berghei CSP after infection with forms. It is interesting to note that even though infected cells did sporozoites and to present the optimal P. berghei CSP CTL epitope not undergo apoptosis, neighboring noninfected cells (probably to specific CD8ϩ T cells. In this light, we show that both infected traversed by sporozoites) readily underwent apoptosis upon addi- and uninfected hepatocytes (traversed by sporozoites) process and tion of specific CTLs (Fig. 7d, third panel). This suggests that the present the CSP, and that processing is largely dependent on pro- CS protein left behind by sporozoites traveling through or between teasomes and, to a lesser extent, aspartic proteases. hepatocytes (27, 46) is presented by traversed hepatocytes. This There is a continuous discussion about the cell type involved finding is further substantiated by the fact that hepatocytes infected in the processing and presentation of the CSP after sporozoite in- with spectϪ/Ϫ sporozoites do not activate T cells as much as those fection. Three cell types may be implicated in this process: Kupffer infected with wt sporozoites, suggesting that traversed hepatocytes cells, DCs, and hepatocytes. process and present the CSP to T cells. This presentation by tra- Although little is known about the role of Kupffer cells, there is versed hepatocytes could represent an escape mechanism elabo- no doubt that DCs can present sporozoite-derived Ags, and a pro- rated by the parasite to evade the host immune system, by directing tective status is attained in mouse experimental models in which a specific T cells to traversed hepatocytes which present malaria- relatively high number of sporozoites are injected (36–38). How- derived peptides. However, further studies are needed to determine ever, in the field, where the number of sporozoites injected via whether this mechanism is operative in vivo. Nevertheless, the fact mosquito bites is low, and with a high proportion of these sporo- that protection could still be observed in Fas- and perforin-defi- zoites going directly to the hepatocyte within minutes (11), DC cient mice (44) suggests that infected cells do not undergo apo- presentation may not be the relevant pathway for priming. Thus, ptosis in vivo. 7062 PRESENTATION OF SPOROZOITE Ags BY HEPATOCYTES

We found that optimum processing and presentation occur 8–20 Disclosures h after infection of hepatocytes with sporozoites (Fig. 1). This is in The authors have no financial conflict of interest. agreement with previous studies conducted in vivo (47). In that ϩ study, it was shown that maximum CD8 T cell activation oc- References curred within the first 8 h after immunization with normal or ir- 1. Clyde, D. F. 1975. Immunization of man against falciparum and vivax malaria by radiated P. yoelii sporozoites, and lasted for ϳ48 h. use of attenuated sporozoites. Am. J. Trop. Med. Hyg. 24: 397–401. The dependence of processing on the proteasome is consistent 2. Nussenzweig, R. S., J. Vanderberg, H. Most, and C. Orton. 1967. Protective immunity produced by the injection of x-irradiated sporozoites of plasmodium with studies which showed that sporozoites deposit the CSP in the berghei. Nature 216: 160–162. cytoplasm of cells, beginning from the time of their attachment to 3. Mueller, A. 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