Genetically Attenuated Parasite Vaccines Induce Contact-Dependent CD8+ T Cell Killing of Plasmodium yoelii Liver Stage-Infected Hepatocytes

Adama Trimnell, Akihide Takagi, Megha Gupta, Thomas L. Richie, Stefan H. Kappe and Ruobing Wang
J Immunol November 1, 2009, 183 (9) 5870-5878; DOI: https://doi.org/10.4049/jimmunol.0900302

Abstract

The production of IFN-γ by CD8+ T cells is an important hallmark of protective immunity induced by irradiation-attenuated sporozoites against malaria. Here, we demonstrate that protracted sterile protection conferred by a Plasmodium yoelii genetically attenuated parasite (PyGAP) vaccine was completely dependent on CD8+ T lymphocytes but only partially dependent on IFN-γ. We used live cell imaging to document that CD8+ CTL from PyGAP-immunized mice directly killed hepatocyte infected with a liver stage parasite. Immunization studies with perforin and IFN-γ knockout mice also indicated that the protection was largely dependent on perforin-mediated effector mechanisms rather than on IFN-γ. This was further supported by our observation that both liver and spleen CD8+ T cells from PyGAP-immunized mice induced massive apoptosis of liver stage-infected hepatocytes in vitro without the release of detectable IFN-γ and TNF-α. Conversely, CD8+ T cells isolated from naive mice that had survived wild-type P. yoelii sporozoite infection targeted mainly sporozoite-traversed and uninfected hepatocytes, revealing an immune evasion strategy that might be used by wild-type parasites to subvert host immune responses during natural infection. However, CTLs from wild-type sporozoite-challenged mice could recognize and kill infected hepatocytes that were pulsed with circumsporozoite protein. Additionally, protection in PyGAP-immunized mice directly correlated with the magnitude of effector memory CD8+ T cells. Our findings implicate CTLs as key immune effectors in a highly protective PyGAP vaccine for malaria and emphasize the critical need to define surrogate markers for correlates of protection, apart from IFN-γ.

Irradiated and genetically attenuated Plasmodium sporozoites (spz)3 are among the most promising pre-erythrocytic malaria vaccine candidates, as both provide complete and long-lasting protection in rodent models of malaria (1, 2, 3, 4, 5, 6, 7). Irradiated spz have been found to induce complete protection in humans (4), and a human trial for a genetically attenunated parasite vaccine is underway as well. The full understanding of the basic immune effector mechanisms that mediate protection in animals immunized with these vaccines will greatly enhance our efforts to design, develop, and evaluate efficacious subunit as well as attenuated sporozoite vaccines against liver stage (LS) malaria.

CD8+ T cells have been found to play a critical role in the induction of protection in mice immunized with radiation-attenuated spz (8, 9). Loss of protective immunity in β2-microglobulin−/− mice and in mice depleted of CD8+ T cells are indicative of the indispensable role of CD8+ T cell-mediated effector mechanisms (10, 11). In studies with the rodent parasites Plasmodium berghei and Plasmodium yoelii, adoptive transfer of CD8+ T cell clones specific for the epitopes expressed in the circumsporozoite proteins (CSP) of the parasites conferred complete protection in naive mice against parasite challenge (12, 13, 14). Protection in humans conferred by irradiated Plasmodium falciparum spz is likewise dependent primarily on the activity of cytotoxic CD8+ lymphocytes (15). Furthermore, CSP-specific CD8+ T cells have been found to efficiently lyse infected hepatocytes in vitro (12, 16, 17). All of these data point toward a requisite role of primed CD8+ T cells in providing sterile protection against spz challenge in the immunized host. However, the T cell-mediated effector mechanisms that inhibit the development of liver stages of malaria parasites remain poorly understood. IFN-γ responses have been correlated with pre-erythrocytic-stage protective immunity for a long time, although the importance of CD8+ T cell-derived IFN-γ, in particular, is not yet fully established (18, 19). Sterile protection obtained with genetically attenuated P. berghei spz was found to correlate quantitatively with IFN-γ-producing CD8+ T cells (3). Protective immunity induced with P. falciparum circumsporozoite protein-based pre-erythrocytic-stage vaccine, RTS,S, was also found to be dependent on IFN-γ-producing CD4+ and CD8+ T cells (20). On the other hand, IFN-γ-independent CD8+ T cell-mediated protective immunity has been demonstrated (21), and Chakravarty et al. recently found that IFN-γ secretion by primed CD8+ T cells is not essential for the protection of mice against live P. yoelii spz challenge (22).

With these contrasting reports explaining the possible effector functions of protective CD8+ T cells in various malaria models, we were intrigued to explore the CD8+ T cell-mediated protective mechanisms contributing to sterile immunity in P. yoelii genetically attenuated parasite (PyGAP)-immunized mice. We had earlier shown that protracted protection mediated by either of two genetic knockout (KO) parasites Pyuis3(−) and Pyuis4(−) was absolutely dependent on CD8+ T cells, whereas depletion of CD4+ T cells did not reverse the protection (5). Here we report that CD8+ T cells from PyGAP-immunized mice led to rapid apoptosis of infected hepatocytes in vitro in an IFN-γ-independent manner. However, PyGAP-induced protection was found to be partly dependent on IFN-γ in immunized mice. Interestingly, PyGAP spz failed to protect against wild-type (wt) spz challenge in most perforin KO mice (PKO), which led us to propose that contact-dependent cytotoxicity by Ag-specific CD8+ T cells is one of the crucial mechanisms leading to rapid clearance of infected hepatocytes in PyGAP-immunized mice, thereby conferring complete sterile protection. PyGAP successfully generated memory responses providing long-term protection, and a population of liver CD8+ T cells in their effector phase (TEM) directly correlated with protective immunity. Our findings have important implications in the design of future T cell-based vaccines against malaria and in the determination of correlates of protection.

Materials and Methods

Mice and parasites

Female 6- to 8-wk-old BALB/c mice and C.129S7(B6)-Ifngtm1Ts/J mice were purchased from The Jackson Laboratory for immunization experiments. Perforin-deficient BALB/c mice (PKO) were provided by Dr. J. T. Harty (University of Iowa). Female 6- to 8-wk-old Swiss Webster (SW) mice purchased from Charles River Laboratories were used for maintenance of P. yoelii parasite life cycle. Animal handling and care were in accordance with Institutional Animal Care and Use Committee-approved protocols. The P. yoelii 17X NL (a nonlethal strain) clone 1.1 Pyuis3(−) and Pyuis4(−) parasites were cycled between Anopheles stephensi mosquitoes and SW mice as previously described (5).

Immunization studies

Groups of BALB/c mice (five mice per group) were immunized by i.v. injections of 50,000 or 10,000 Pyuis3(−) or Pyuis4(−) spz via the tail vein. Booster injections were administered 2 wk apart. Immunized mice were challenged 14 days postimmunization by i.v. injection with 10,000 wt P. yoelii spz.

Separate sets of immunization experiments were conducted with genetic KO mice. PKO and IFN-γ KO mice in a BALB/c background were injected with 50,000 Pyuis4(−) spz i.v. via tail vein. Immunized mice were boosted 2 wk apart.

Wild-type P. yoelii challenges

All immunized mice were challenged with 10,000 wt P. yoelli spz after 7 days of immunization(s). Protection was determined by the absence of parasites in Giemsa-stained blood smears taken daily from day 2 to day 6 postchallenge.

Naive BALB/c mice, as a nonprotective model, were also challenged at the same time with a single dose of 10,000 wt P. yoelli spz and the parasitemia levels were checked thereafter. Splenocytes and liver-infiltrated lymphocytes were used as controls for cellular assays from these mice 4 wk after the challenge.

CD8+ T cell and IFN-γ depletion studies

CD8+ T cells and IFN-γ were depleted in immunized mice. They were injected i.p. daily with 0.5 mg per dose of anti-CD8 mAb 2.43 (TIB210; American Type Culture Collection) for 4 consecutive days before challenge. For depletion of IFN-γ, mice were administered 1 mg per dose of mAb XMG-6 (anti-IFN-γ) for 4 consecutive days before challenge (23). They were administered anti-IFN-γ mAb on the day of challenge and 3 days postchallenge as well. All treated and naive control mice were challenged with 10,000 wt spz 14 days after the last immunization. Control mice received equivalent doses of control rat IgG. These doses and regimens were found to be optimal for >95% depletion efficiency of CD8+ T cells and IFN-γ in mice (data not shown).

Cellular inhibition of LS development assay

Primary hepatocytes were isolated from BALB/c mouse as previously described except for the use of a steady-state manual liver perfusion instead of a mechanical pump (24). Isolated hepatocytes were diluted and dispensed at 100,000 cells in 250 μl per well in Lab-Tek chamber coverslip slides or 40,000 cells in 100 μl per well in 96-well tissue culture plates (Nunc) and incubated overnight at 37°C in an atmosphere of 5% CO2 in air. The next day, hepatocyte cultures were infected with 80,000 GFP-tagged P. yoelii wt spz in 100 μl of medium per chamber well and 30,000 in 50 μl of medium per well of 96-well plate. Infected cultures were further incubated for 16 h at 37°C in an atmosphere of 5% CO2 in air. Each well was washed three times with 300 μl of Eagle’s complete MEM to remove unattached spz. Fresh medium plus dexamethasone was added thereafter to each well and cultures were incubated for 8 h at 37°C in an atmosphere of 5% CO2 in air for hepatic stage development. The infected hepatocytes were then exposed to enriched splenic or intrahepatic CD8+ T cells from immunized or control mice. To introduce physical separation between lymphocytes and infected hepatocyte population, intrahepatic lymphocytes were incubated with infected hepatocytes in presence or absence of filter in a 96-transwell plate (Corning).

Enriched CD8+ T cells from liver and spleen were isolated 5 days postimmunization of mice as previously described (23). Lymphocytes were also isolated from naive mice 4 wk postchallenge with wt P. yoelli spz. Splenocytes were isolated from sacrificed mice by passing spleens through a 70-μm cell strainer (BD Biosciences), and erythrocytes were lysed with RBC lysis buffer (eBioscience). Liver cells were collected by passing the liver tissue through a 100-μm cell strainer, and leukocytes were separated by Percoll gradient centrifugation (Sigma-Aldrich) followed by lysis of erythrocytes. Spleen and liver CD8+ T cells from these leukocyte fractions were purified using the MACS system negative selection beads according to the manufacturer’s instructions (Miltenyi Biotec). Twenty million splenocytes and 2 million liver infiltrate cells were used to purify and negatively select CD8+ T cell populations.

In certain experiments, infected hepatocytes were pretreated with 2 μg/ml PyCSP peptide (SYVPSAEQI; purchased from Sigma-Aldrich at >95% purity) or with ECFp control pool of 23 peptides from human influenza virus (purchased from SynPep at >90% purity) for 4 h at 37°C before coculturing with 50,000 enriched spleen or liver CD8+ T cells per chamber per well for 16 h. Peptide-stimulated infected cultures were thoroughly washed with complete medium before adding spleen or liver CD8+ T cells. Live imaging or apoptosis assays were performed on these cultures.

In vitro live cell imaging

To study live cellular interactions, liver- or spleen-enriched CD8+ T cells from immunized or control mice were loaded with the lysomotrophic stain LysoTracker Red (Invitrogen), according to the manufacturer’s recommendations. These enriched CD8+ T cells were added to primary hepatocyte cultures infected with GFP-tagged wt P. yoelii spz,3 and images were captured using a Delta Vision microscope (Applied Precision). Samples were recorded at 2-min intervals in multiple z-layers with a double wavelength excitation of 488 nm for GFP expression by LS-infected hepatocytes and 535 nm for LysoTracker Red; differential interference contrast images for each z-plane and time frame were recorded and data were analyzed using the softWoRx software (Applied Precision). Relative distance traveled by each CD8+ T cell toward the LS-infected hepatocyte was assessed per sample.

Apoptosis assay and indirect immunofluorescence

At 48 h after spz invasion and coculturing with spleen/liver-enriched CD8+ T cells, live apoptosis assays were performed using enzymatic assays for caspase-3 and caspase-7 according to the manufacturer’s recommendations (FLICA Red from Immunochemistry Technologies). FLICA Red reagent that permeabilizes the cells and deposit a red fluorescent compound at the site of enzyme activity was used to detect CTL-induced apoptosis of the hepatocytes. For the enzymatic detection assay, 0.8 μl of 1/5 dilution of the FLICA Red reagent was added in 100 μl each of supernatant per well of adherent LS-infected hepatocyte culture and incubated for 40 min at 37°C, 5% CO2. Samples were washed five times each with 1× FLICA Red wash buffer or PBS. This was followed immediately by indirect immunofluorescence assay to stain LS parasites in the culture. After the final wash step for each apoptosis assay, samples were blocked with 5% BSA in PBS followed by incubation with anti-MAP4 (anti-CSP/Hep17) Ab or NYLS3 mAb (for Hep17) at room temperature for 1 h. After five washes in PBS, samples were incubated for 45 min at room temperature with the anti-rabbit IgG Alexa Fluor 488-conjugated Ab at a dilution of 1/1000 in 2.5% BSA in PBS. Samples were then washed five times with PBS. To detect apoptotic nuclei, samples were then treated with the Hoechst stain (as recommended by the manufacturer, Immunochemistry Technologies). Treated samples were viewed using a fluorescence-inverted microscope (Eclipse TE2000-E; Nikon) and images were recorded and analyzed using the MetaMorph Office version 7.0 software.

Results

GAP-induced protection is partially dependent on IFN-γ and perforin

In protected models of irradiated spz, CD8+ T cell-derived IFN-γ has been found to be responsible for complete protection (17, 19, 25). To determine whether such an effector mechanism plays an important role in PyGAP-induced protection, groups of BALB/c mice (n = 5/group) were immunized with three doses of 10,000 or 50,000 of Pyuis3(−) or Pyuis4(−) spz as previously described (5). CD8+ T cells or IFN-γ were depleted in immunized mice before challenging them with 10,000 wt P. yoelii spz.

In conformity with our previous report, all immunized mice depleted of CD8+ T cells developed patent blood stage infections 1 day later than did nonimmunized control mice (Fig. 1⇓). Immunized mice that received IgG as a control remained completely protected. Half (50%) of the IFN-γ-depleted mice that were immunized with 3 × 10,000 Pyuis4(−) spz, 3 × 50,000 Pyuis4(−) spz, or 3 × 50,000 Pyuis3(−) spz were completely protected (Fig. 1⇓). This suggests that both Pyuis3(−) and Pyuis4(−)-induced protection is partially dependent on IFN-γ. However, immunization with 10,000 Pyuis3(−) spz led to parasitemia in all of the mice after IFN-γ depletion, unlike those immunized with 10,000 Pyuis4(−) spz. This is likely explained by the fact that Pyuis4(−) spz confers more robust protection as compared with Pyuis3(−) spz at low doses of immunization. Therefore, IFN-γ depletion might cause increased failure of protection in mice immunized with low dose of Pyuis3(−) spz.

FIGURE 1.
FIGURE 1.

GAP-induced protection is completely dependent on CD8+ T cells and partially dependent on IFN-γ. Mice immunized with Pyuis4(−) (A and B) or Pyuis3(−) (C and D) spz were injected with anti-CD8+ Ab or anti-IFN-γ Ab before challenge as described in Materials and Methods. Control mice received equal amounts of control IgG Ab. Graphs show the percentage of protected mice after challenge with 10,000 wt P. yoelii spz. Protection was determined by parasitemia in depleted mice from day 2 to day 6 after challenge. The data are representative of two independent experiments.

Immunization of IFN-γ ΚΟ mice and subsequent challenge with wt P. yoelii spz also led to protection of 50% mice from parasitemia in two independent experiments (Fig. 2⇓), further reiterating our finding that IFN-γ plays a partial role in providing protection to the immunized mice.

FIGURE 2.
FIGURE 2.

GAP-induced protection is dependent partly on IFN-γ and perforin. BALB/c mice, IFN-γ KO mice, and PKO mice in groups of five each were immunized with three doses of 50,000 Pyuis4(−) spz as described in Materials and Methods. Immunized mice were challenged with 10,000 wt P. yoelii spz 14 days postimmunization. Protection was determined by checking parasitemia in mice from day 2 to day 6 after challenge. The data are representative of two separate experiments.

To probe the importance of perforin-mediated killing by CD8+ T cells in PyGAP-induced immunity, PKO mice were immunized with 3 × 50,000 Pyuis4(−) spz. It was found that at the end of 6 days, only 20% of immunized PKO mice were protected from the wt challenge (Fig. 2⇑), indicating a partial role of perforin in the conferment of protection by PyGAP spz.

In contrast, the naive mice that were challenged with 10,000 wt spz were 100% infected. Parasitemia levels reached up to 50–60% ∼7–10 days after challenge but were cleared out completely by 3 wk postchallenge.

CD8+ T cell mediates contact-dependent killing of LS-infected hepatocytes

These in vivo depletion studies indicated that PyGAP-induced protection is completely dependent on CD8+ T cells but only partially dependent on IFN-γ. This prompted us to further investigate if cytolytic activity of CD8+ T cells mediates the protection by killing LS-infected hepatocytes and whether CD8+ T cell-derived IFN-γ is involved in the process. Primary hepatocytes infected with GFP-tagged wt P. yoelii spz (26) were cocultured with CD8+ T cells isolated from mice immunized with three doses of Pyuis4(−) and loaded with fluorescent lysomotrophic probe LysoTracker Red. Fig. 3⇓ and supplemental video 14 show the timeframe snapshots and live video of interaction between the target LS-infected hepatoctyte and LysoTracker-stained Pyuis4(−)-induced CD8+ T cells, respectively, over a period of 1200 s at 120-s intervals, starting from 0 s after the addition of the CD8+ T cells. As seen in Fig. 3⇓A, immediately after adding the Pyuis4(−)-specific CD8+ T cells into the infected hepatocyte culture (red arrows), one LysoTracker-stained T cell settled directly above the infected hepatocyte (green arrow) and another unstained T cell moved toward the infected hepatocyte membrane (yellow arrow). Through a time course of 0–960 s, the infected hepatocyte cell membrane and LS parasite residing inside became larger in size and, subsequently, green fluorescence of the GFP-tagged LS parasite gradually diminished, indicating the death of both the parasite and the infected hepatocyte. The LysoTracker Red stain was rapidly released from the loaded CD8+ T cells and taken up by the targeted hepatocyte, suggesting the mobilization of lytic granules. At the 840-s time point after the CTL action, the T cell moved away from the LS-infected hepatocyte cell membrane (Fig. 3⇓C, 840 s). In contrast, the CD8+ T cells from a mock control mouse that was immunized with mosquito salivary gland debris did not migrate toward the LS-infected hepatocyte, even after a time course of 1200 s (Fig. 3⇓D and video not shown). This indicated that Pyuis4(−) CD8+ T cells specific for P. yoelii LS Ags recognized the infected hepatocytes and led to the cytotoxicity.

FIGURE 3.
FIGURE 3.

PyGAP-specific CD8+ T cells lyse infected hepatocytes. Primary mouse hepatocytes were infected with GFP-tagged wt P. yoelii spz. Twenty-four hours postinfection, CD8+ T cells from a PyGAP-immunized mouse or control mouse loaded with LysoTracker Red were added to the infected cultures. Live cellular interactions were monitored under the DeltaVision fluorescence microscope. A–C, Immediately after adding PyGAP-specific CD8+ cells (red arrows) into the LS parasite (green arrows)-infected hepatocyte culture, one T cell was already on top and another one was next to the membrane of the infected hepatocyte (yellow arrows). Through the time course (A, 0–1200 s), the infected heptocyte cell membrane and the LS parasite appeared swollen and LysoTracker Red stained contents dispersed in the infected hepatocyte. The T cell started moving away from the damaged hepatocyte at 840 s. D, CD8+ T cells from a control mouse that has received debris of noninfected mosquitoes did not migrate toward the infected hepatocyte even after 20 min of observation. These data are representative of three separate experiments.

To verify the movement of primed CD8+ T cells specifically toward infected hepatocytes, relative distance traveled by CD8+ T cells toward or away from the LS-infected hepatocyte was measured. Fig. 4⇓ shows the overall dynamics of movement of PyGAP-specific spleen CD8+ T cells toward LS-infected hepatocyte at 24 h (Fig. 4⇓A) and 48 h (Fig. 4⇓B) post invasion with P. yoelii GFP-tagged spz. It shows that spleen CD8+ T cells from Pyuis4(−) immunized mice are specifically attracted to LS-infected hepatocyte as compared with the nonspecific random movements of T cells from mock control or naive mice.

FIGURE 4.
FIGURE 4.

Kinetics of PyGAP-specific spleen CD8+ T cell movement toward LS-infected hepatocyte. Primary mouse hepatocytes were infected with GFP-tagged wt spz and at 24 h and 48 h post infection, spleen CD8+ T cells from a Pyuis4(−) immunized mouse, mock control-immunized mouse, or naive mouse that had been loaded with LysoTracker Red were individually added to the infected cultures. Movement of CD8+ T cells was observed and recorded under the DeltaVision fluorescence microscope and data were analyzed using the softWoRx software. A, The relative distance traveled by each CTL through a time course of 0–960 s at 24 h incubation. B, The relative distance traveled by each CTL through a time course of 0–600 s at 48 h incubation. These data represent three separate experiments, and the average of each CTL’s relative distance either toward or away from the LS-infected hepatocyte was recorded as positive (+) or negative (−) on the graph.

PyGAP-specific CD8+ T cells mediate apoptosis of infected hepatocytes

To ascertain the pathways leading to death in CTL-targeted infected hepatocytes, live enzymatic apoptotic assays were performed. Cocultures of CD8+ T cells and infected primary hepatocytes were stained to detect the classical apoptosis markers: caspase-3 and caspase-7. Sequentially, the cultures were stained with polyclonal anti-MAP4 Abs (stains CSP and Hep17) to detect the LS-infected hepatocytes. Both spleen and liver CD8+ T cells from Pyuis4(−)-immunized mice induced apoptosis of the infected hepatocytes (Fig. 5⇓A–C). This was confirmed by the presence of numerous apoptotic bodies (white arrows) and fragmented nuclei (stained blue). Apoptotic LS-infected cells appeared detached and shrunken with the presence of apoptotic bodies. Fig. 5⇓C shows an apoptotic cell harboring LS parasite next to an apoptotic nucleus. Despite membrane blebbing, the cells did not lose the membrane integrity. which confirms the occurrence of apoptosis rather than necrosis. In contrast, spleen or liver CD8+ T cells from mock control mice did not trigger apoptosis of the infected hepatocytes (Fig. 5⇓, D and E). Few background apoptotic cells were observed, possibly due to mechanical shearing while preparing the primary hepatocytes, but most of the infected cells were seen intact. Thus, physical contact of Pyuis4(−) CTLs with infected hepatocytes initiated a cascade of events that led to rapid apoptosis of infected cells. A general observation that the LS parasites (green) in the infected hepatocytes cocultured with Pyuis4(−) T cells (Fig. 5⇓A) appeared smaller in size than those from samples incubated with mock control T cells (Fig. 5⇓D) suggests that CD8+ T cells from Pyuis4(−)-immunized mice may also induce inhibition of parasite development inside the liver cells. These results give credence to the live imaging observations of PyGAP CD8+ T cell-induced contact-dependent killing of infected hepatocytes (supplemental video 1).

FIGURE 5.
FIGURE 5.

PyGAP-specific CD8+ T cell-induced apoptosis of LS-infected hepatocytes. Primary hepatocytes were cultured in chambered glass coverslips and infected with wt P. yoelii spz. At 24 h post infection, intrahepatic (A and B) or spleen (C) CD8+ T cells from mice immunized with three doses of 10,000 Pyuis4(−) spz or mice that had been immunized with mosquito salivary gland debris (D and E) were added to the infected cultures. Forty-eight hours after spz invasion and coculturing with the T cells, the liver cells were stained with FLICA Red for the enzymes caspase-3 and caspase-7, followed immediately by staining with anti-MAP4 (anti-CSP/Hep17) polyclonal Ab and rabbit IgG Alexa Fluor 594 Ab as described in Materials and Methods. Images were monitored using the fluorescence microscope. LS parasites were stained green, apoptotic cells were stained red, and apoptotic nuclei were stained blue.

Surprisingly, none of the common cytokines (IFN-γ or TNF-α) was detected in the supernatants of the cultures from the infected hepatocyte in the presence of Pyuis4(−)-specific CTLs, as measured by ELISA (data not shown) or the ultrasensitive electrochemiluminescence MSD mouse multiplex cytokine assay (Fig. 6⇓A). This shows that apoptotic activity of Pyuis4(−) CTLs leading to the clearance of LS parasites was independent of the secretion of IFN-γ. Results obtained by physically separating the lymphocytes and infected hepatocyte in cocultures by transwell experiment also reiterated the fact that killing by CD8+ T cells was indeed contact dependent and cytokines do not play a major role in cytotoxicity. There was an ∼24% decrease in the number of LS parasites in wells where PyGAP-specific lymphocytes were physically separated from infected hepatocytes, whereas we observed almost 85% inhibition of LS parasites in wells without filters (Fig. 6⇓B). Nevertheless, it does not rule out the possibility that there is potentiation of CD8+ T cell activity by IFN-γ in vivo.

FIGURE 6.
FIGURE 6.

Secretion of cytokines by CD8+ T cells during contact-dependent killing of infected hepatocytes. A, Electrochemiluminescence MSD mouse multiplex cytokine assay was used to detect IFN-γ, TNF-α, and IL-2 in picogram quantities in supernatants of the cultures from the infected hepatocyte cultures treated with Pyuis4(−) CTLs. B, Intrahepatic lymphocytes from Pyuis4(−)-immunized mice or mock-immunized mice were added to infected cultures in 96-well plate in presence or absence of filter to separate cell populations. Forty-eight hours after spz invasion, cultures were stained with anti-MAP4 (anti-CSP/Hep17) polyclonal Ab and rabbit IgG Alexa Fluor 594 Ab as described in Materials and Methods. LS parasites were counted under the fluorescence microscope. Experiment was performed in triplicates.

Wild-type spz-induced CTLs trigger apoptosis of noninfected hepatocytes

To understand the basis of protection in PyGAP-immunized mice more clearly, we compared the activity of CD8+ T cells from a nonprotective model (nonimmunized mice that recovered from 10,000 wt P. yoelii spz challenge) with the activity of CD8+ T cells from a protecitve model (Pyuis4(−)-immunized mice). Strikingly, the CD8+ T cells from the wt spz-challenged mice did not recognize the infected cells but indeed caused apoptosis of surrounding uninfected cells (Fig. 7⇓A), contrasting with Pyuis4(−) CTL activity that completely eliminated all of the infected cells (Fig. 7⇓B). It has been shown in various studies that the surrounding uninfected cells are traversed by the spz before infecting a single hepatocyte (27, 28). While traversing, spz shed their surface protein, circumsporozoites in the traversed cells, which is processed and presented with MHC class I on the surface of hepatocytes (16, 28). Therefore, it is likely that the CD8+ T cells from the wt spz-challenged mice mainly recognize CSP on the surface of uninfected traversed hepatocytes. To confirm this, the infected cell cultures were pulsed with CSP CTL epitope before coincubating with the CD8+ T cells. There was no significant change in the activity of Pyuis4(−) CTLs even when added to the CSP-pulsed cultures of infected hepatocytes. Massive apoptosis of infected as well as uninfected hepatocytes was observed (Fig. 7⇓D). In case of CD8+ T cells from wt-challenged mice, there was an increase in the apoptosis of infected cells along with the death of uninfected cells (Fig. 7⇓C). The infected cells were not recognized by these wt spz-primed CD8+ T cells when LS-infected cultures were treated with a control peptide (Fig. 7⇓E). These results strongly suggest that most CD8+ T cells from wt spz-challenged mice were specific for CSP epitope and failed to recognize other LS Ags expressed on the surface of infected hepatocytes. Thus, more likely, non-CSP LS Ag-specific CTLs induced by GAP immunization play a crucial role in the complete elimination of LS parasites.

FIGURE 7.
FIGURE 7.

Cellular inhibition of LS parasites in presence of CD8+ T cells from PyGAP-immunized mice and from wt-challenged mice. CD8+ T cells from mouse that had recovered from challenge with a single dose of 10,000 wt P. yoelii spz (A) or intrahepatic CD8+ T cells from mice immunized with three doses of 10,000 Pyuis4(−) spz (B) were cocultured with infected primary hepatocytes in a 96-well plate as described in Materials and Methods. Forty-eight hours later, cultures were sequentially treated with FLICA Red to detect caspase-3 and caspase-7 followed by staining with anti-Hep17 mAb (NYLS3). Staining with Hoechst reagent was done to detect apoptotic bodies. Stained slides were observed under inverted fluorescence microscope for the presence of apoptotic cells (stained red), LS parasites (stained green), and apoptotic nuclei (stained blue). In another experiment, infected hepatocytes were pretreated with 2 μg/ml PyCSP peptide (SYVPSAEQI) for 4 h at 37°C before coculturing with enriched spleen or liver CD8+ T cells from wt-challenged mice (C) or PyGAP-immunized mice (D). Infected hepatocytes stimulated with control pepetide ECFp and subsequently treated with CD8+ T cells from wt-challenged mice were used as controls (E). LS parasites (stained green) in primary hepatocytes were manually counted per sample using the fluorescence microscope (F). Scale bar, 100 μm. Values shown are the mean (±SD) values from four independent experiments.

CTL-mediated killing of infected hepatocytes correlate with protective immunity in PyGAP-immunized mice

We wanted to determine whether the PyGAP-induced CTL activity against LS-infected hepatocytes in vitro correlates with the protection against spz challenge in vivo. LS-infected hepatocyte cultures were stained with mAb against LS specific Ag (Hep17) and quantified at the end of 48 h of CTL assay to assess the percentage inhibition of LS parasites. There were on average 31 LS parasites per well after incubation with T cells from liver of the mock control mouse that received noninfected mosquito debris, as compared with 24 and 6 LS parasites per well after coculturing with T cells from mice that recovered from wt spz challenge and mice immunized with Pyuis4(−) spz, respectively (Fig. 7⇑F). The percentage inhibition of LS parasite was 22 and 80 for CD8+ T cells for wt spz-challenged and PyGAP-immunized mice, respectively. To determine the cytolytic activity induced by CSP-specific T cells, LS-infected hepatocyte cultures were sensitized with the CSP-derived CTL epitopes before initiating CTL assay. The cellular inhibition of LS development by CD8+ T cells from the wt spz-challenged or PyGAP-immunized mice was increased to 66% and 92%, respectively. A similar pattern of inhibition was observed for CD8+ T cells obtained from the spleen (Fig. 7⇑F). The PyGAP-induced CTL killing of LS-infected hepatocytes was significantly greater than that induced by CTLs from wt spz-challenged mice (CSP specific, p = 0.002 and non-CSP specific, p = 0.0009; Student’s t test). However, there was a 44% increase of the inhibition of LS-infected hepatocytes in the presence of CSP peptide in the cultures, indicating that the CTL killing of infected hepatocytes by the CTLs derived from P. yoelii wt spz challenge was mainly CSP-specific. Nevertheless, the in vitro killing data for CD8+ T cells from PyGAP-immunized mice was correlated with the protective immunity induced by PyGAP spz in vivo.

Increase of liver TEM cells is a potential indicator of protective immunity

The importance of memory T cell responses for long-term protection has been described for malaria vaccines (29, 30, 31). TEM were detected in mice immunized with P. berghei irradiated-spz and PbGAP vaccines (3, 32). Here, we investigated the generation of memory T cell phenotypes in PyGAP-immunized mice. Substantial populations of TEM and central memory T cells (TCM) were detected in spleen and liver of immunized mice. We correlated these T cell populations with protection induced after each dose of immunization with Pyuis4(−) as compared with that in control mice that received salivary gland lysate from noninfected mosquitoes. Five days after primary immunization with 10,000 Pyuis4(−) spz, there was decline in liver CD8+ TCM and proportional increase in TEM cells, whereas TCM and TEM pools in spleen were comparable with those in control mice (Table I⇓). However, those immunized mice were not protected when challenged with 10,000 wt spz on day 7 after primary immunization (Table I⇓), indicating that the magnitude of TEM cells was insufficient to mount protective immunity.

View this table:
Table I.

PCI: ratio of TEM/TCM correlated with protection induced by GAP Pyuis4(−) immunization

Following a larger primary immunization dose of 50,000 Pyuis4(−) spz, which resulted in complete protection, we observed an increased differentiation of TCM cells to TEM cells in both liver and spleen (Table I⇑). This was seen by CD62L down-regulation and CD44 up-regulation of CD8+ T cells 5 days after immunization. The increase in the ratio of TEM to TCM was more prominent in the liver than in the spleen. There was no further increase in the ratio of the T cell memory phenotypes when boosted with 50,000 Pyuis4(−) spz, indicating that the magnitude of TEM cells may have reached its maximum level after a single high-dose immunization. Thus, quantifying the ratio of TEM to TCM, termed the protection correlation index (PCI), may be a viable measure of protective immunity.

Discussion

The focus of our study was to elucidate the CD8+ T cell-mediated effector mechanisms that contribute to the sterile immunity induced by PyGAPs, Pyuis4(−), and Pyuis3(−) in BALB/c mice. Previously, we reported that CD8+ T cell responses were indispensable for PyGAP-induced protracted sterile protection (5). Apart from the release of IFN-γ, other potential effector mechanisms deployed by CD8+ T cells have not been explored thoroughly. To the best of our knowledge, this is the first report that provides direct evidence of contact-dependent cytotoxicity as one of the important methods used by GAP-specific CD8+ T cells to eliminate LS parasites. Partial loss of protection in PKO and IFN-γ KO mice indicated that PyGAP-induced protection is partly dependent on IFN-γ as well as perforin. However, noticeably, CD8+ T cell-mediated cytotoxicity of infected hepatocytes in vitro was found to be independent of IFN-γ release.

Live imaging studies of time-lapse microscopy using enriched liver or spleen CD8+ T cells from Pyuis4(−)-immunized mice showed rapid movement of CD8+ T cells toward hepatocytes infected with P. yoelii spz. Swelling of the hepatocyte membrane was observed as CD8+ T cells made direct contact and dispersed the lysomotrophic probe into the infected cells, indicating the discharge of lytic granules. Caspase-3 and caspase-7 staining suggested apoptosis of infected target hepatocytes. This was similar to that reported in detail by Wiedemann et al. demonstrating the polarization of lytic granules of CTL toward the target cell (33). The signaling molecules that are involved in specific movement of PyGAP-specific spleen CD8+ T cells, however, remain to be elucidated. Lyubchenko et al. reported that calcium influx is involved at the point of contact for granule exocytosis (34). More detailed studies are needed to determine the precise requirements for granule exocytosis in PyGAP-induced CTL activity. Another recent study also described similar contact-dependent apoptotic activity of CSP-specific CD4+ T cell clones (35). CD8+ T cell-mediated lytic activity would require the recognition of processed liver stage Ags in complex with MHC class I on the surface of infected hepatocytes. It is quite likely that professional APCs like dendritic cells may be involved during initial priming of CD8+ T cells in the immunized mice as discussed elsewhere (36, 37, 38). The fact that spleen-derived CD8+ T cells also exhibited rapid lysis of infected hepatocytes suggests that such immune-responsive tissues can be potential sites for CD8+ T cell priming, as the parasite traverses multiple tissues and sheds its surface proteins before invading the liver (39). Another study supports a similar notion where the authors observed that the first cohort of CD8+ T cells is primed in cutaneous lymph nodes, which subsequently travels to the liver during malaria infection (36). IFN-γ and other inflammatory cytokines released during immunization or infection can possibly direct the CTLs to the site of action. It is noteworthy that the in vitro apoptotic activity of CD8+ T cells was independent of CD8+ T cell-derived IFN-γ, but the likelihood of potentiation of the CTL activity by IFN-γ from other sources in vivo cannot be ruled out. Our finding corroborates recent studies that found protection induced by CD8+ T cells against LS parasite was independent of CD8+ T cell-derived IFN-γ (22). Our in vivo studies, however, indicated that IFN-γ was partially required for the generation of protective immunity in PyGAP-immunized BALB/c mice. Apart from direct cytotoxic effects on infected cells, IFN-γ may also be involved in complex cellular interactions facilitating the activation of immune cells, including CD8+ T cells. IFN-γ has been used as a surrogate marker for immunity in evaluating potential of vaccines against LS malaria infection (20, 40, 41, 42). Our findings warrant the search for more definite correlates of protective immunity in addition to this pleiotropic cytokine.

Interestingly, the liver CD8+ T cells from naive mouse that survived wt P. yoelii spz (10,000 spz) challenge did not kill the infected hepatocytes in vitro. On the contrary, they recognized and lysed the surrounding noninfected hepatocytes. This may be indicative of an immune evasion strategy adopted by wt spz to divert the host immune system toward uninfected cells. This idea has been proposed previously but no evidence has been put forward until now (43). This kind of condition is likely to operate among the populations that live in malaria endemic areas, thereby explaining the absence of protective immune responses against the infection even after repeated exposures to the parasite. It has also been shown earlier that infected cells are protected from apoptosis by hepatocyte growth factor/MET signaling (44, 45). Our results are also supported by the findings of Bongfen et al., who demonstrated that CSP, an immunodominant spz surface protein, is processed and presented by both traversed and infected primary hepatocytes and that they were both efficiently lysed by CSP-specific CTLs, apparently via perforin/granzyme mobilization (16). Since in vitro cell traversal and hepatocyte invasion are very similar between wt and GAP spz (our unpublished data), it can be very well envisaged that in wt infection, since the infected cells are protected from apoptosis (46) and thus the presentation of various LS Ags is inhibited (37), most of the CD8+ T cell population from wt spz-challenged mice is primed to CSP, which is presented by traversed hepatocytes. In contrast, Pyuis4(−)-induced CD8 T cells rapidly recognize infected hepatocytes in addition to traversed hepatocytes. However, Pyuis4(−)-specific CD8 T cells are capable of eliminating both spz-traversed and LS parasite-infected hepatocytes after incubation of infected hepatocyte cultures with CSP-specific CTL epitope peptide. Hypothetically, growth-arrested GAP spz break down early in their development in the hepatocyte and stimulate immune responses against a plethora of LS Ags in the immunized mice (5). As a consequence, heterogeneous mixtures of Ag-specific CTL responses against infected hepatocytes are much more vigorously induced by GAP immunization than are the CSP-specific CTL responses induced by wt spz infection.

Recently, Belnoue et al. demonstrated that live P. yoelii infected erythrocytes under chloroquine treatment conferred protection not only against challenge by blood stage parasites but also against sporozoite challenge, indicating that a shared repertoire of Ags in liver and blood stage parasites might be responsible for the protection (47). Similarly, CD8+ T cells from PyGAP-immunized mice might also recognize various LS Ags apart from CSP presented on the infected primary hepatocytes. Another line of evidence that gives credence to our hypothesis is the cross-species protection conferred by P36p-deficient P. berghei spz to P. yoelii LS parasites (48). Induction of T cell responses against other LS Ags other than CSP contribute to cross-species protective immunity since the circumsporozoite proteins of P. berghei and P. yoelii do not share any common T cell epitopes. In support of our data, we found that CSP peptide pulsing of infected hepatocyte culture led to rapid apoptosis of both the infected and traversed cells by the CD8+ T cells from wt spz-challenged mice. This fundamental difference in the activity of PyGAP CTLs when compared with that of CTLs from wt spz-challenged mice correlates with the protection observed in PyGAP-immunized mice.

Apart from CTL-mediated cytotoxicity, rapid generation of TEM was also found to be associated with protection in PyGAP-immunized mice. We defined the ratio of TEM to TCM as the protection correlation index (PCI). The kinetics of memory CD8+ T cell activation has been shown to be similar in liver and spleen in earlier reports of LS malaria infection (49). Here, we demonstrate that a TEM population of memory T cells was effective in mounting a protective immune response, and high PCI in liver correlated with protection observed in BALB/c mice after immunization with a high dose (50,000 spz) of Pyuis4(−). The absence of protection with a lower dose (10,000 spz) was in agreement with the low PCI observed. A relevant study by Schmidt et al. (31) recently defined a threshold frequency of memory CD8+ T cells in blood that predict long-term sterilizing immunity against LS infection. Our report is in agreement with that of Hafalla et al. that a single priming dose can induce memory T cell effector responses sufficient enough to render complete protection (49). This can prove to be critical for future considerations of malaria LS vaccine design in terms of immunization protocols for optimal efficiency.

In summary, we identified CD8+ T cell-mediated contact-dependent killing of infected hepatocytes as one of the important effector mechanism in PyGAP-BALB/c model of protection against LS malaria. Along with IFN-γ, cell-mediated killing should be considered an important criterion in identifying new LS vaccine candidates. Our in vitro assay to quantify cellular inhibition of LS development in presence of CD8+ T cells has the potential to be employed as a method for selection of LS Ags targeted by T cells for vaccine development. Determination of PCI can also prove to be an important surrogate marker for protection in vivo. Therefore, in the face of new challenges met in identifying potential candidates for malaria vaccine, we need to redefine the parameters that correlate with protection.

Acknowledgments

We thank Dr. Amy de Rocher at Seattle Biomedical Research Institute for her assistance with the Delta Vision microscopy. PKO mice were provided by Dr. John T. Harty (University of Iowa).

Disclosures

The authors have no financial conflicts of interest.

Footnotes

  • The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

  • ↵1 This work was supported by a M. J. Murdock Trust and Seattle Biomedical Research Institute innovation grant.

  • ↵2 Address correspondence and reprint requests to Dr. Ruobing Wang, 307 Westlake Avenue N, Suite 500, Seattle Biomedical Research Institute, Seattle, WA 98109. E-mail address: ruobing.wang{at}sbri.org

  • ↵3 Abbreviations used in this paper: spz, sporozoite; GAP, genetically attenuated parasite; KO, knockout; LS, liver stage; PCI, protection correlation index; PKO, perforin knockout; PyGAP, Plasmodium yoelii genetically attenuated parasite; TCM, central memory T cell; TEM, effector memory T cell; wt, wild type; CSP, circumsporozoite protein.

  • ↵4 The online version of this article contains supplemental material.

  • Received February 9, 2009.
  • Accepted August 24, 2009.

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