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The Journal of Immunology, 2008, 181, 1420 -1428
Copyright © 2008 by The American Association of Immunologists, Inc.
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Malaria-Specific and Nonspecific Activation of CD8+ T Cells during Blood Stage of Plasmodium berghei Infection1

Mana Miyakoda*, Daisuke Kimura*, Masao Yuda{ddagger}, Yasuo Chinzei{ddagger}, Yoshisada Shibata{dagger}, Kiri Honma* and Katsuyuki Yui2,*

* Division of Immunology, Department of Molecular Microbiology and Immunology, and {dagger} Atomic Bomb Disease Institute, Graduate School of Biomedical Sciences, Nagasaki University, Nagasaki, Japan; and {ddagger} Department of Medical Zoology, School of Medicine, Mie University, Tsu, Japan


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Cerebral malaria is one of the severe complications of Plasmodium falciparum infection. Studies using a rodent model of Plasmodium berghei ANKA infection established that CD8+ T cells are involved in the pathogenesis of cerebral malaria. However, it is unclear whether and how Plasmodium-specific CD8+ T cells can be activated during the erythrocyte stage of malaria infection. We generated recombinant Plasmodium berghei ANKA expressing OVA (OVA-PbA) to investigate the parasite-specific T cell responses during malaria infection. Using this model system, we demonstrate two types of CD8+ T cell activations during the infection with malaria parasite. Ag (OVA)-specific CD8+ T cells were activated by TAP-dependent cross-presentation during infection with OVA-PbA leading to their expression of an activation phenotype and granzyme B and the development to functional CTL. These highly activated CD8+ T cells were preferentially sequestered in the brain, although it was unclear whether these cells were involved in the pathogenesis of cerebral malaria. Activation of OVA-specific CD8+ T cells in RAG2 knockout TCR-transgenic mice during infection with OVA-PbA did not have a protective role but rather was pathogenic to the host as shown by their higher parasitemia and earlier death when compared with RAG2 knockout mice. The OVA-specific CD8+ T cells, however, were also activated during infection with wild-type parasites in an Ag-nonspecific manner, although the levels of activation were much lower. This nonspecific activation occurred in a TAP-independent manner, appeared to require NK cells, and was not by itself pathogenic to the host.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Malaria remains one of the crucial threats to public health in much of the world. It has been well accepted that Ab and CD4+ T cells play critical roles for protection against malaria parasites that can be acquired during natural or experimental infection (1, 2, 3, 4). However, the role of CD8+ T cells in protective immunity is controversial. Some studies suggested that CD8+ T cells could transfer protective immunity to an adoptive host (5), whereas others claimed that they did not play a major role in protection against blood stage infection with Plasmodium species (6). In contrast, accumulating evidence indicates that CD8+ T cells are involved in the pathogenesis of severe malaria. Cerebral malaria resulting from Plasmodium falciparum infection is one of the most severe complications and main cause of death in human malaria (7, 8, 9, 10, 11). Using a rodent model of malaria infection with the Plasmodium berghei ANKA (PbA)3 strain, investigations indicated that CD8+ T cells are one of the major effector cells that trigger cerebral malaria. In these experimental models, it was shown that CD8+ T cells were sequestered in the brain during cerebral malaria and that the depletion of CD8+ T cells decreased mortality (8). Furthermore, perforin-mediated killing by CD8+ T cells was required for the pathogenesis of experimental cerebral malaria resulting from PbA infection, suggesting that the effector function of CD8+ T cells is involved in the pathogenesis (9). However, it is unclear whether Plasmodium-specific CD8+ T cells are activated during the erythrocyte stage of malaria and how they are involved in the pathogenesis of cerebral malaria.

Because malaria parasites infect RBC that do not themselves express MHC molecules, the infected cells cannot directly present malaria Ags to T cells in association with MHC class I molecules. To activate specific CD8+ T cells, malaria Ags must be presented by a process referred to as cross-presentation by APCs that are themselves not infected, as reported for some minor histocompatibility Ags, tumor Ags, and various pathogens (12, 13). There are two main Ag presentation pathways of cross-presentation; one is the phagosome-to-cytosol pathway that is dependent on the TAP molecule (14), and the other is the TAP-independent pathway in which antigenic peptide is generated and loaded to MHC class I in MHC class II compartments (15) or on the cell surface by peptide regurgitation (16). The former TAP-dependent pathway is used by viruses that do not themselves infect hemopoietic cells (17) or some of the intracellular parasites such as Mycobacterium tuberculosis and Toxoplasma gondii (18, 19). The latter TAP-independent pathway is used by some pathogen-derived Ags such as those from Leishmania major and virus-like particles (20, 21). It is unclear how these different pathways are selected in targeting the exogenous Ags to the MHC class I presentation pathway by each microbial species.

Because class I-restricted Ags have not been identified in malaria parasites during the erythrocyte stage, we used a model Ag OVA to study immune responses of Ag-specific CD8+ T cells during malaria infection. We generated a recombinant PbA that expresses a cytoplasmic form of OVA (OVA-PbA). Using this system, we show that malaria Ags can be presented to specific CD8+ T cells by cross-presentation in a TAP-dependent manner during the erythrocyte stage of malaria infection and that these activated CD8+ T cells could be pathogenic to the host. Furthermore, CD8+ T cells that are not specific for malaria Ag can be activated during malaria infection in an Ag-nonspecific manner. This activation was at least in part dependent upon the presence of NK cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Generation of recombinant PbA parasite clones expressing OVA

Recombinant PbA parasites were engineered to constitutively express a truncated C-terminal fragment of OVA (aa 150–386) fused to the N-terminal sequence (aa 1–5) of the PbA heat shock protein (hsp) 70 gene. The gene construct was based on pBluescript KS+ (Stratagene) and contains the following elements: 1) PbA dihydrofolate reductase-thymidyltransferase-ts (DHFR-ts) gene; 2) PbA hsp70 5'-untranslated region and N-terminal coding sequence; 3) coding sequence of C-terminal fragment of OVA; 4) PbA hsp70 3'-untranslated region and DHFR-ts 3'-untranslated region. The DHFR-ts gene contains a point mutation at position 110 of the DHFR gene causing a Ser->Asn transition conferring resistance to the antimalaria drug pyrimethamine.

The procedure to generate recombinant PbA was described previously (22). In brief, the gene construct was digested with SacI and KpnI to linearize and release the insert from the vector. PbA merozoites were transfected by electroporation and were selected in rats using pyrimethamine. The surviving parasites were further selected by limiting dilution in mice, and parasite clones that were resistant to pyrimethamine were obtained.

Mice, adoptive transfer, and PbA infection

OT-I-transgenic mice expressing the TCR specific for OVA257–264/Kb (23), were provided by Dr. H. Kosaka (Osaka University, Osaka, Japan), TAP knockout (TAP-KO) mice (C57BL/6 (B6) background; Ref. 24) by Dr. H. Watanabe (University of the Ryukyus Okinawa, Japan), B6.SJL-Ptprc congenic (B6.SJL) mice (CD45.1+) by Dr. Y. Takahama (University of Tokushima, Tokushima, Japan), and RAG2 knockout (RAG2-KO) mice (25) by Dr. Y. Yoshikai (Kyushu University, Fukuoka, Japan). TCR P14 lymphocytic choriomeningitis virus (LCMV)/TCR{alpha}-KO mice (26) were purchased from Taconic. B6 mice were purchased from SLC. OT-I and B6.SJL mice were bred, and offspring were intercrossed to obtain CD45.1 OT-I mice. RAG2-KO mice and OT-I mice were intercrossed to obtain RAG2-KO OT-I mice. These mice were maintained in the Laboratory Animal Center for Animal Research at Nagasaki University (Nagasaki, Japan) and were used at the age of 8–14 wk. For adoptive transfer, CD8+ T cells (>95%) were purified from CD45.1 OT-I mice using anti-CD8 IMag (BD Biosciences), labeled with CFSE (15 µM; Molecular Probes) and were injected into the tail vein of B6 mice (0.7–2 x 107/mice). Mice were infected with WT-PbA or OVA-PbA by i.p. injection of parasitized RBCs (104 infected RBC) or by i.v. injection of 106 parasitized RBC (Fig. 2C). Parasitemia was monitored by microscopic examination of standard blood films, and mice were sacrificed after the parasitemia reached 1.6–18.2% (days 7–9). Brain sequestered lymphocytes were prepared as described (27). Depletion of NK cells was performed by injection of anti-NK1.1 mAb (PK136; 50 µl of ascites fluid partially purified by ammonium sulfate precipitation method) i.p. at –1 day, and i.v. at 2, 5, and 7 days after infection with PbA. The animal experiments reported herein were approved by the Institutional Animal Care and Use Committee of Nagasaki University and were conducted according to the guidelines for Animal Experimentation at Nagasaki University.

Immunoblotting

Peripheral blood cells of PbA-infected mice were washed and then lysed in PBS by freezing and thawing. After centrifugation, lysate (107 RBC) was separated on 15% SDS-PAGE and transferred to polyvinylidene difluoride membrane. The blot was blocked and probed with rabbit anti-OVA (Bethyl Laboratories) or rabbit anti-merozoite surface protein 1 Ab. After a washing, the membrane was incubated with HRP-conjugated anti-rabbit Ig Ab, washed, and analyzed using ECL reagents.

Proliferation and IFN-{gamma} production in vitro

To isolate dendritic cells (DC), B6 spleen was treated with dispase (5 µg/ml; Godo-shusei) for 30 min at 37°C. Spleen cells were treated with anti-CD11c microbeads, and DCs (CD11c+, >90%) were purified using Auto MACS (Miltenyi Biotech). Proliferation of OT-I was determined by culture of OT-I CD8+ T cells (1 x 105) in the presence of DC (3 x 103) and crude RBC Ag (9.3 x 106 RBCs) for 63 h with [3H]TdR for the final 15 h. For IFN-{gamma} assay, OT-I CD8+ T cells (3 x 105) were stimulated with DCs (1 x 104) in the presence of crude malaria Ag (9.3 x 106 RBC) for 48 h, and the levels of IFN-{gamma} in the supernatant were determined by a sandwich ELISA as described previously (28).

Flow cytometry

FcR was blocked with anti-Fc{gamma}RII/III mAb (BD Biosciences). The staining reagents used in this study include PE-Cy7-anti-CD45.1, allophycocyanin- or PE-anti-CD8, FITC-anti-V{alpha}2, PE- or FITC-anti-CD62 ligand (CD62L), FITC- or biotin-anti-CD44, and PE- or FITC-anti-CD69 mAbs (eBiosciences). OVA257–264 H-2Kb tetramer was purchased from MBL. 7-Aminoactinomycin D was added to exclude dead cells from the analysis. For analysis of granzyme B expression, splenocytes were incubated with Fc block and were stained with PE-Cy7-anti-CD45.1 and FITC- or allophycocyanin-anti-CD8 mAbs. Samples were fixed and permeabilized using Cytofix-Cytoperm buffer (BD Biosciences), stained with PE-anti-granzyme B mAb (Caltag), and analyzed using FACSCanto II (BD Biosciences).

Cytotoxicity assay in vitro and in vivo

For in vitro cytotoxicity assay, CD8+ T cells were enriched from spleen cells by CD8a+ T cell isolation kit (Miltenyi Biotech). EL4 target cells were labeled with 51Cr with and without OVA257–264 peptide (OVAp) for 1 h at 37°C. Target and effector cells were added to each well of a 96-well plate and cultured for 4 h. Percent specific lysis = [(experimental 51Cr release – spontaneous 51Cr release)/(maximum 51Cr release – spontaneous 51Cr release)] x 100. For in vivo cytotoxicity assay, B6 spleen cells were labeled with a high concentration of CFSE (10 µM) and were pulsed with OVAp (1 µg/ml) for 1 h at 37°C or labeled with low concentration of CFSE (1 µM) and were incubated without peptide. Cells from each population were mixed at a 1:1 ratio, and a total of 1 x 107 cells were adoptively transferred into the recipient mice. Mice were sacrificed 4 h later, and spleen cells were analyzed using flow cytometry. Percent specific lysis = [1 – (ratio of peptide-pulsed cells)/(ratio of peptide-unpulsed cells)] x 100.

Evaluation of the disease

Mice were monitored daily after day 5 of infection, and clinical scores were defined by the presence of the typical pathological signs as described (29): level 1, hunching or wobbly; level 2, hunching and wobbly; level 3, hypotonia; level 4, limb paralysis and convulsions; level 5, death.

Statistical analysis

In comparison of three or more groups, overall comparison was first made by ANOVA for one-way or two-way data at the significance level of 0.05, and if significant, each pair of the groups was compared by t test. If the ordinary ANOVA was considered to be inappropriate because of the significant departure from normality, censoring in measurements or large variation in the variance among groups, the Kruskal-Wallis test, the Wilcoxon rank-sum test, or the Savage test was used instead with the significance level determined by the Bonferroni procedure controlling the familywise error rate <0.05. For survival data, the log-rank test was used in a similar way. Procedures of ANOVA, LIFETEST, and NPAR1WAY in the SAS system were used for the calculations.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Ag-specific and nonspecific activation of CD8+ T cells during PbA infection

We generated recombinant malaria PbA parasite that constitutively expresses the C-terminal fragment of OVA (aa 150–386) fused to the N terminus of PbA hsp70 (OVA-PbA; Fig. 1A). The expression of the recombinant OVA was confirmed by immunoblotting of the infected RBC lysates with anti-OVA Ab (Fig. 1B). The ability of DCs to present OVA-PbA Ag was evaluated for proliferation and IFN-{gamma} secretion of OVA-specific OT-I CD8+ T cells in vitro (Fig. 1, C and D). CD8+ T cells from OT-I mice showed specific proliferation and IFN-{gamma} secretion in response to the DCs pulsed with OVA-PbA-infected RBC. We used adoptive transfer of OT-I CD8+ T cells from TCR-transgenic mice to determine whether Ag-specific CD8+ T cells can be primed during PbA infection. B6 mice (CD45.2+) were adoptively transferred with CFSE-labeled OT-I CD8+ T cells (CD45.1+) and were infected with wild-type P. berghei ANKA (WT-PbA) or OVA-PbA. The proliferation of OT-I CD8+ T cells was monitored by the sequential loss of CFSE intensity 7 days after infection (Fig. 2, A and B). The proportion of OT-I CD8+ T cells increased in mice infected with OVA-PbA (19.4%) when compared with uninfected mice (4.2%) or mice infected with WT-PbA (3.1%). OT-I CD8+ T cells divided minimally and remained CD69–CD44lowCD62Lhigh in the uninfected host. Extensive division of OT-I CD8+ T cells was observed in mice infected with OVA-PbA. In addition, up-regulation of the CD69 marker was observed in cells that divided 0–3 times, and up-regulation of CD44 and down-regulation of CD62L were evident in cells that have divided multiple times, indicating that OT-I CD8+ T cells were activated in an Ag-specific manner during infection with PbA. Unexpectedly, OT-I CD8+ T cells divided ~4 times and up-regulated the CD69 marker in mice infected with WT-PbA, suggesting that OT-I CD8+ T cells can be activated in an Ag-nonspecific manner during infection with WT-PbA. Because it was recently reported that malaria infection impairs cross-presentation (30), we examined the ability of host APC to present OVA-PbA Ag during early (days 0–3) and late (days 5–8) periods after infection (Figs. 2C and 4A, left). In both periods, OT-I cells proliferated several times in vivo after infection with OVA-PbA but not with WT-PbA, indicating that APC can cross-present malaria Ag throughout infection with PbA.


Figure 1
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  		FIGURE 1. The expression of OVA in recombinant PbA. A, DNA construct to generate recombinant PbA. The C-terminal OVA coding sequence (aa 150–386) was fused to the N terminus (aa 1–5) of the PbA hsp70 gene. The expression of OVA was controlled by the PbA hsp70 promoter, which is constitutively active during liver and blood stages of the malaria lifecycle. DHFR-ts is a selection marker, which confers resistance to the antimalaria drug pyrimethamine. B, The lysates of RBC from B6 mice infected with WT-PbA or OVA-PbA were separated by SDS-PAGE, blotted, and probed with anti-OVA Ab and with anti-merozoite surface protein 1 Ab as control. C, OT-I T cells (3 x 105) and splenic DCs (CD11c+ cells, 1 x 104) were cultured in the presence of uninfected freeze-thaw lysates of 9.3 x 106 RBCs or those infected with WT-PbA or OVA-PbA for 2 days, and pulsed for 20 h with [3H]TdR. Parasitemia levels: WT-PbA, 16.9%; OVA-PbA, 18.4%. D, OT-I spleen cells (1 x 105) were cultured in the presence of DCs (CD11c+ cells, 3 x 103) and RBCs from uninfected mice ({square}) or mice infected with WT-PbA ({blacktriangleup}) or OVA-PbA (•) for 48 h. The production of IFN-{gamma} in the supernatant was determined by ELISA.

 

Figure 2
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  		FIGURE 2. Ag-specific and -nonspecific activation of OT-I CD8+ T cells in vivo during PbA infection. A, CFSE-labeled OT-I CD8+ T cells (CD45.1+, 8.8 x 106) were adoptively transferred into B6 mice, and the mice were uninfected or infected with WT-PbA or OVA-PbA (1 x 104) i.p. on the same day. Seven days later, spleen cells were stained with allophycocyanin-anti-CD8, PE-Cy7-anti-CD45.1, and PE-anti-activation markers (CD69, CD44, CD62L). Staining profiles of activation markers and CFSE are shown for the CD45.1+CD8+ gated population. Numbers in the upper right corner indicate percent of cells above the line. Levels of parasitemia: WT-PbA. 10.0%; OVA-PbA, 8.4%. B, Summary of cell divisions in the spleen after adoptive transfer of CFSE-CD8+ T cells (top). Data were obtained from four independent experiments similar to A. Bottom, Percentage of CD69+CD8+ T cells obtained from six independent experiments similar to A except that transferred OT-I cells were not labeled with CFSE and the staining was performed using FITC-anti-CD69 mAb. In both panels, three groups showed a significant difference in overall comparison (p < 0.05; top, Savage test; bottom, Kruskal-Wallis test); each bar and whisker denote the mean and SD, respectively. The difference in the number of cell divisions was slightly not significant between uninfected and WT-PbA-infected mice (p = 0.0187, Savage test), whereas it was significant between uninfected and OVA-PbA-infected mice (p = 0.0046), and between WT-PbA-infected and OVA-PbA-infected mice (p = 0.0047). The proportion of CD69+CD8+ T cells was significantly higher in WT-PbA-infected (p = 0.0025, Wilcoxon rank-sum test) and OVA-PbA-infected mice (p = 0.0025) when compared with uninfected mice, whereas it was not significant (p = 0.1490) between WT-PbA-infected and OVA-PbA-infected mice. C, CFSE-labeled OT-I CD8+ T cells (CD45.1+, 1 x 107) were adoptively transferred into B6 mice, which were uninfected or infected with the large dose (1 x 106) of WT-PbA or OVA-PbA i.v. Three days later, spleen cells were analyzed using flow cytometry. Levels of parasitemia: WT-PbA, 1.3%; OVA-PbA, 1.0%.

 
We have also analyzed lymphocytes in the brain. The number of CD8+ T cells retained within the brain was increased in mice infected with WT-PbA or OVA-PbA (Fig. 3A). A large variation in the number of CD8+ T cells in the infected mice might reflect their differential stages of cerebral malaria. OT-I cells, however, were increased only in mice infected with OVA-PbA. The number of CD4+ T cells was not significantly different between these groups of mice. We also examine the CFSE profile and surface phenotype of OT-I cells that were sequestered in the brain (Fig. 3B). OT-I cells in the brain of OVA-PbA-infected mice uniformly showed low levels of CFSE and CD44highCD62Llow phenotype, indicating that only highly activated OT-I cells were retained within the brain of mice infected with OVA-PbA.


Figure 3
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  		FIGURE 3. Sequestration of T cells in the brain of mice infected with PbA. A, B6 mice were adoptively transferred with OT-I CD8+ T cells (CD45.1+) and were uninfected or infected with WT-PbA or OVA-PbA. After elevation of parasitemia (WT-PbA, 2.2–12.9%; OVA-PbA, 1.2–8.4%), lymphocytes in the brain were collected and stained with allophycocyanin-anti-CD8, PE-anti-CD4, and PE-Cy7-anti-CD45.1 mAb. The numbers of T cell subsets were determined by multiplying the total number of cells with the percentage of each population. Overall comparison showed a significant difference both in the number of CD8+ T cells and in OT-1 (p < 0.05, Kruskal-Wallis). However, the difference in the former was slightly not significant between uninfected and WT-PbA-infected mice (p = 0.0179, Wilcoxon rank-sum test) and between uninfected and OVA-PbA-infected mice (p = 0.0328), whereas in the latter the difference was significant between OVA-PbA-infected mice and WT-PbA-infected mice (p = 0.0112) and was slightly not significant between OVA-PbA-infected mice and uninfected mice (p = 0.0328). Overall comparison of the number of CD4 showed no significant difference (p = 0.1394, Kruskal-Wallis test). B, CFSE-labeled OT-I CD8+ T cells (CD45.1+) were adoptively transferred into B6 mice, which were uninfected or infected with WT- or OVA-PbA. Brain lymphocytes were stained with mAbs as described in Fig. 2A. Staining profiles of activation markers and CFSE are shown for the CD45.1+CD8+ gated populations. Numbers in the upper right corner indicate percentage of cells above the line. Levels of parasitemia: WT-PbA, 10.1%; OVA-PbA, 9.4%. Representative data of two similar results are shown.

 
Activation of malaria-specific CD8+ T cells is TAP dependent

To determine whether the Ag presentation of OVA epitope to OT-I CD8+ T cells utilize the conventional class I Ag presentation pathway, we examined the requirement for the TAP molecule in proliferation of OT-I CD8+ T cells in vivo in TAP null mice (TAP-KO; Fig. 4, A and C). In this experimental system, we monitored the T cell response in vivo within 3 days after transfer, given that the number of OT-I CD8+ T cells was severely reduced 5 days after transfer, perhaps due to their rejection in TAP-KO host mice (data not shown). Thus, we infected B6 or TAP-KO mice with WT-PbA or OVA-PbA, adoptively transferred CFSE-labeled CD8+ T cells from CD45.1+OT-I mice 5 days later, and monitored proliferation of OT-I T cells by the sequential loss of CFSE intensity 3 days later (parasitemia 2.3–5.4%). Division of OT-I cells was not detectable in uninfected or WT-PbA-infected hosts during these 3 days. In B6 mice infected with OVA-PbA, however, OT-I cells divided several times, indicating that they were activated in vivo in an Ag-specific manner. In TAP-KO hosts, no proliferative responses were observed, indicating that Ag presentation of the OVA epitope expressed in OVA-PbA in association with the MHC class I molecule, was dependent on the TAP molecule. However, OT-I CD8+ T cells showed increased expression of CD69 and reduced expression of CD62L in the TAP-KO host mice infected with WT-PbA or OVA-PbA, suggesting that these phenotypical changes of OT-I CD8+ T cells could be induced without TCR occupancy during PbA infection (Fig. 4, B and C). We also evaluated their expression of granzyme B by intracellular staining in this model. The expression of granzyme B was undetectable in OT-I CD8+ T cells in uninfected mice but was detected in OT-I CD8+ T cells in WT-PbA or OVA-PbA-infected mice both before and after culture (Fig. 4, D and E). The level of granzyme B expression in OT-I CD8+ T cells ex vivo was higher in OVA-PbA-infected mice than in WT-PbA-infected mice. OT-I CD8+ T cells transferred into TAP-KO mice also showed up-regulation of granzyme B expression, suggesting that it could be induced without TCR occupancy during malaria infection.


Figure 4
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  		FIGURE 4. Specific activation of OT-I CD8+ T cells during infection with OVA-PbA is TAP dependent. B6 or TAP-KO mice were uninfected or infected with WT-PbA or OVA-PbA. Five days later, mice were inoculated with CFSE-labeled CD8+ T cells (1 x 107 in A, 2 x 107 in B) from CD45.1+OT-I mice. Three days later, spleen cells were stained with allophycocyanin-anti-CD8, with PE-Cy7-anti-CD45.1 (A), or with additional FITC-antiactivation marker mAbs (CD69, CD44, CD62L) (B). The CFSE profiles (A) or staining profiles of activation markers (solid line) and isotype control (dark histogram) (B) of the CD45.1+CD8+ gated populations are shown. C, Summary of the cell divisions (n = 3) and CD69+ cells (n = 2; each dot represents the result of one mouse) performed as in A and B. In B6 mice, a significant difference was observed in overall comparison of the number of cell divisions among three groups of mice (p < 0.05, Savage test), whereas no such difference was observed in TAP-KO mice. In B6 mice, the number of cell division was significantly larger in OVA-PbA-infected mice compared with WT-PbA-infected mice (p = 0.0143, Savage test) and uninfected mice (p = 0.0127), while the difference was not significant between WT-PbA-infected and uninfected (p = 0.0732); each bar and whisker denote the mean and SD, respectively. D and E, Staining profiles of the CD45.1+CD8+ gated populations of spleen cells prepared as in B, stained with allophycocyanin-anti-CD8, PE-Cy7-anti-CD45.1, and PE-anti-granzyme B (Gzm B) mAbs or PE-isotype control ex vivo before (D) and after culture on plates coated with anti-TCR mAb for 5 h (E). The number in each panel indicates the percentage of positive cells. The high background of the isotype control (23.9%) was observed without PE-isotype Ab, suggesting that it was due to autofluorescence. Levels of parasitemia in WT-PbA (3.2–5.4%) and OVA-PbA (2.4–6.1%)-infected B6 mice: WT-PbA (2.3–6.2%)-infected mice; and OVA-PbA (4.7–5.5%)-infected TAP-KO mice.

 
Involvement of NK cells in nonspecific activation of CD8+ T cells

It has been reported that NK cells are activated during malaria infection and play pivotal roles in the induction and recruitment of specific CD8+ T cells (31, 32, 33). To examine whether CD4+ T cells, other CD8+ T cells, or NK cells are involved in the activation of OT-I CD8+ T cells, we infected RAG2-KO OT-I mice, which lack an adaptive immune system except for monoclonal OVA-specific OT-I CD8+ T cells, with PbA. OT-I CD8+ T cells showed clear up-regulation of the CD69 marker in mice infected with WT-PbA or OVA-PbA, indicating that OT-I CD8+ T cells did not require CD4+ T cells or other CD8+ T cells for their activation (Fig. 5A). The expression of granzyme B was also detected in OT-I CD8+ T cells in RAG2-KO OT-I mice that were infected with WT-PbA or OVA-PbA, indicating that the help of CD4+ T cells or other CD8+ T cells was not required for the induction of granzyme B (Fig. 5B). When NK cells were depleted by treatment with anti-NK1.1 mAb in vivo, up-regulation of CD69 and the expression of granzyme B were severely impaired in CD8+ T cells from WT-PbA-infected mice (Fig. 5). OT-I CD8+ T cells, however, were activated in NK1.1-treated OVA-PbA-infected mice at levels indistinguishable from the control group, indicating that NK cells are not required for Ag-specific activation of CD8+ T cells during blood stage infection with PbA. It was reported that NK markers are expressed in T cells of virus-infected mice (34). In RAG2-KO OT-I mice, ~14 and 21% of CD69+CD8+ T cells became NK1.1+ during infection with WT-PbA and OVA-PbA, respectively (data not shown). Therefore, the effect of NK1.1 mAb on WT-PbA-infected mice was not simply due to the direct depletion of activated CD8+ T cells. In addition, the reduction of the activated OT-I CD8+ T cells was seen in WT-PbA-infected mice and not in OVA-PbA-infected mice. Taken together, these results suggested that NK cells were involved in Ag-nonspecific activation of CD8+ T cells during PbA infection.


Figure 5
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  		FIGURE 5. Involvement of NK cells in the nonspecific activation of OT-I CD8+ T cells during infection with PbA. RAG2-KO OT-I mice were inoculated with PBS or anti-NK1.1 mAb on –1, 2, 5, and 7 days after infection with WT-PbA or OVA-PbA. On day 8, spleen cells were stained with PE-anti-CD8 and FITC-labeled activation markers (CD69, CD62L, CD44; solid line; A), or with FITC-anti-CD8 and PE-anti-granzyme B (Gzm B; B). Data represent staining profiles of CD8+-gated populations. Levels of parasitemia: WT-PbA, PBS-treated (6.5%), NK-depleted (5.4%); OVA-PbA, PBS-treated (18.2%), NK-depleted (14.2%). Values are representative data of two similar results.

 
To examine whether nonspecific activation occurs in CD8+ T cells expressing other TCR during malaria infection, we used CD8+ T cells from P14-transgenic mice, which express TCR specific for LCMV (26). P14 CD8+ T cells up-regulated CD69, down-regulated CD62L, and expressed granzyme B after infection with WT-PbA, indicating that activation of CD8+ T cells during PbA infection is not limited to OT-I cells (Fig. 6).


Figure 6
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  		FIGURE 6. Nonspecific activation of CD8+ T cells from P14 TCR-transgenic mice. Eight days after infection with WT-PbA, spleen cells from P14 mice were stained with anti-CD8 and FITC-labeled activation markers (CD69, CD62L, CD44) or PE-anti-granzyme B (Gzm B). Data represent staining profiles of CD69, CD62L, and CD44 (solid line) and granzyme B of CD8+-gated populations. The level of parasitemia was 15.7%.

 
CTL function of T cells activated by malaria infection

To determine whether OT-I CD8+ T cells that are activated during PbA infection are able to kill targets, we performed CTL assays in vitro. B6 mice were transferred with OT-I CD8+ T cells and were infected with WT-PbA or OVA-PbA. CD8+ T cells were enriched from these mice and were subjected to 51Cr release assay (Fig. 7A). OT-I CD8+ T cells in OVA-PbA-infected B6 mice showed specific CTL activity against OVA-pulsed targets. OT-I CD8+ T cells from WT-PbA-infected mice showed weak but significant OVA-specific killing activity. We also examined CTL activity of CD8+ T cells in RAG2-KO OT-I mice. These cells showed OVA-specific CTL activity after infection with WT-PbA or OVA-PbA, indicating that CTL can be induced without help of CD4+ or other CD8+ T cells (Fig. 7B). The CTL activity of OT-I CD8+ T cells from OVA-PbA-infected mice was much higher than those from WT-PbA-infected mice, consistent with their higher expression of granzyme B (Fig. 5B).


Figure 7
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  		FIGURE 7. CTL activity in vitro of CD8+ T cells during PbA-infection. A, B6 mice were uninfected or infected with WT-PbA or OVA-PbA. Five days later, mice were inoculated with CD8+ T cells (1.1 x 107) from CD45.1+OT-I mice. Three days later, CD8+ T cells were purified by negative selection (>83%) and were subjected to 51Cr release assay using OVAp-pulsed (1 µg/ml; •) and unpulsed ({circ}) EL4 targets for 4 h. Proportions of OT-I cells in CD8+ T cells: uninfected, 8.0%; WT-PbA, 11.0%; OVA-PbA, 31.1%. Levels of parasitemia: WT-PbA, 3.4%; OVA-PbA, 4.0%. B, RAG2-KO OT-I mice were infected with WT-PbA or OVA-PbA. Eight days later, purified CD8+T cells were subjected to 51Cr release assay as in A. The number of OT-I cells in CD8+T cells was determined based on the percent of OVAp/H-2Kb tetramer-positive cells. Levels of parasitemia: WT-PbA, 5.9%; OVA-PbA, 5.6%.

 
We also evaluated in vivo killing activity of CD8+ T cells during malaria infection. B6 mice were transferred or not transferred with OT-I CD8+ T cells and were infected with WT-PbA or OVA-PbA. Seven days after the infection, these mice received splenocytes that were differentially labeled with CFSE and were left unpulsed (CFSElow) or pulsed (CFSEhigh) with OVAp. The spleen cells were analyzed 4 h later (Fig. 8). OVAp-coated target cells were specifically and almost completely cleared within 4 h in OT-I CD8+ T cell-transferred OVA-PbA-infected mice. Peptide-pulsed targets were also significantly reduced in OT-I CD8+ T cell-transferred WT-PbA-infected mice, although the levels of reduction were much less than those in OVA-PbA-infected mice. These effects were not seen in OT-I CD8+ T cell-transferred uninfected mice or in the infected mice without OT-I CD8+ T cell transfer.


Figure 8
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  		FIGURE 8. Cytotoxicity in vivo of OT-I CD8+ T cells during PbA infection. A, B6 mice were uninfected or infected with WT-PbA or OVA-PbA and were inoculated with lymphocytes from RAG2-KO OT-I mice. Seven days later, mice received a 1:1 mixture of differentially CFSE-labeled target splenocytes (1 x 107), and the cytotoxicity was determined 4 h after target cell transfer. Numbers in plots represent the ratio of peptide-pulsed or unpulsed cells. Proportions of OT-I cells in CD8+ T cells: uninfected, 2.7%; WT-PbA, 5.0%; OVA-PbA, 30.7%. Levels of parasitemia: WT-PbA (1.6%) or OVA-PbA (9.7%)-infected B6 mice with OT-I; WT-PbA (8.7%) or OVA-PbA (10.5%)-infected B6 mice without OT-I. B, Summary of percent-specific lysis of three similar in vivo cytotoxicity experiments. Result of the experiment in A (•) and two other similar experiments using B6 mice transferred with OT-I CD8+ T cells ({square}, {circ}) are shown. The variation among experiments was not significant in each group of mice (p = 0.97, ANOVA for two-way layout data); hence the data in each group of mice were pooled to compare the difference in percent specific lysis; a significant difference was observed in both overall and paired comparison (p < 0.05, ANOVA for one-way layout data).

 
Pathogenesis of CD8+ T cells activated by malaria infection

To determine the role of CD8+ T cells activated during malaria infection, B6, RAG2-KO, and RAG2-KO OT-I mice were infected with WT- or OVA-PbA (Fig. 9). B6 mice died 8–12 days after infection with WT- or OVA-PbA with clinical signs of cerebral malaria. Although the incidence of the cerebral malaria in B6 mice was relatively low (60–80%), it was within the range reported by Amani et al. (35). RAG2-KO mice did not develop cerebral malaria and survived >30 days after infection with WT- or OVA-PbA, consistent with previous studies indicating the requirement for CD8+ T cells in the development of cerebral malaria (7, 8, 9, 10, 11). RAG2-KO OT-I mice were resistant to WT-PbA infection, similar to RAG2-KO mice, and survived >30 days after infection, suggesting that nonspecific activation of CD8+ T cells is not by itself harmful to the host. However, RAG2-KO OT-I mice showed levels of parasitemia higher than RAG-2 KO mice and three of five mice died 13–29 days after infection with OVA-PbA. Although statistical analysis of these data showed that the difference in survival time was not significant between RAG2-KO OT-I and RAG2-KO mice in this particular experiment, we think that it is likely due to the small number of the mice used in this experiment. We observed similar data in another set of experiments; six of seven RAG-2 KO OT-I mice died 15–29 days after infection with OVA-PbA, whereas none of RAG-2 KO mice died within 30 days after infection. Taken together, these data suggest that the activation of malaria-specific CD8+ T cells, in the absence of a diverse adaptive immune system, could lead to the development of lethal pathogenesis during infection with blood stage PbA.


Figure 9
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  		FIGURE 9. Involvement of CD8+ T cell activation in the pathogenesis of PbA. B6 ({circ}), RAG-2 KO (x), and RAG-2 KO OT-I ({square}) mice were infected with WT-PbA (left) or OVA-PbA (right). A, Kaplan-Meier estimation of survival distributions of mice infected with WT- or OVA-PbA. In WT-PbA-infected mice, survival time in B6 mice was significantly shorter than in other two groups (p = 0.0018, log-rank test). In OVA-PbA-infected mice, survival time in B6 mice was significantly shorter than in RAG-2KO OT-I (p = 0.0165) and in RAG-2 KO (p = 0.0014), whereas the difference in survival time was not significant between RAG-2KO OT-I and RAG-2 KO (p = 0.0494). Clinical scores (B) and levels of parasitemia (C) were also determined in each group after infection. Representative data of two similar results are shown.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
This study indicated using a model malaria Ag, OVA, that malaria Ag can be presented to specific CD8+ T cells by APCs in TAP-dependent cross-presentation during infection with PbA. The requirement of TAP for this pathway suggests that cross-presentation of malaria Ags involves the phagosome-to-cytosol pathway, in which Ags are exported to the cytosol after engulfment and are transported into the ER via TAP molecules, as reported for some other microorganisms such as M. tuberculosis and T. gondii (12, 13, 18, 19). Although infection with the malaria parasite might modulate the function of DCs or inhibit cross-presentation (30, 36, 37, 38), our study clearly indicates that APCs are able to cross-present malaria Ags that they have engulfed and activate specific CD8+ T cells during the erythrocyte stage of malaria infection. Although we used a model Ag, OVA, it is likely that the endogenous malaria Ags are presented in a similar manner. The identification of natural CTL epitopes expressed in the erythrocyte stage of malaria parasites would aid our understanding of the role of CD8+ T cells against the malaria blood stage.

In addition to Ag-specific response of CD8+ T cells, we have found that Ag-nonspecific CD8+ T cells could proliferate, show activation phenotype, express granzyme B, and gain CTL function when the host mice were infected with PbA, albeit at a lower level. A couple of possibilities might account for this nonspecific activation of OT-I CD8+ T cells. First, OT-I CD8+ T cells might directly recognize the PbA epitope by cross-reactivity of their TCR. We think that this possibility is unlikely, because OT-1 CD8+ T cells were activated not only in B6, but also in TAP-KO hosts, which are defective in the phagosome-to-cytosol pathway of Ag presentation, suggesting that the activation of OT-I CD8+ T cells in vivo by WT-PbA did not require TCR engagement. In addition, a similar activation-phenotype was observed in CD8+ T cells of P14 TCR-transgenic mice as well as other RAG2-KO TCR-transgenic mice during infection with malaria parasites (Fig. 6 and unpublished observations). Second, host CD8+ T cells might be activated by parasite products via interaction with their receptors other than TCR. Naive and activated CD8+ T cells express a variety of pathogen-recognizing receptors including TLRs (39). Engagement of these receptors with ligands derived from parasites might modulate T cell function without TCR signaling. In particular, it is known that TLR2 is expressed on activated T cells and exhibits costimulatory function for TCR-stimulated T cells or can directly induce Th1 effector function (40, 41). Malaria parasites express GPI anchors that are recognized by TLR2 (42), thus possibly directly modulating the function of host T cells. However, the activation of naive CD8+ T cells by TLR stimulation has not been reported. A third possibility is that CD8+ T cells are activated by cytokine(s) produced by the innate immune system in response to PbA infection (43, 44, 45). Our study suggested that NK cells are involved in nonspecific activation of CD8+ T cells. NK cells produce cytokines such as IFN-{gamma} and TNF-{alpha} during malaria infection (31). Naive T cells can be activated by cytokines without TCR engagement, which has been termed the innate T cell activation pathway (46). Taken together, it is likely that cytokines produced by NK cells, in combination with products of malaria parasites, participate in Ag-nonspecific activation of CD8+ T cells during infection with PbA.

We demonstrated that two types of CD8+ T cells are activated during malaria infection: those specific for malaria Ag and activated by TAP-dependent Ag presentation; and those activated nonspecifically. In both types of activation, CD8+ T cells express the activation phenotype and granzyme B and can develop into functional CTL, although the levels of the nonspecific activation are much lower than the specific activation. Our study suggested that CD8+ T cells that are activated in an Ag-specific manner are involved in the pathogenesis of severe malaria. Highly activated OT-I CD8+ T cells preferentially sequestered in the brain of B6 mice that were transferred with OT-I cells and infected with OVA-PbA (Fig. 3). In this experiment, however, it was unclear whether these cells were involved in the pathogenesis of cerebral malaria, since host B6 CD8+ T cells were sufficient to cause cerebral malaria. In contrast, RAG2-KO OT-I mice that were infected with OVA-PbA showed early death when compared with RAG2-KO mice, suggesting that activation of OT-I CD8+ T cells was pathogenic to the host, likely due to bystander mechanisms (Fig. 9). OVA-PbA-infected RAG2-KO OT-I mice showed more severe parasitemia and died later than B6 mice, suggesting that the death of RAG2-KO mice was not caused by cerebral malaria but may have been caused by other pathological processes associated with the infection. Taken together, these results suggest that the activation of malaria-specific CD8+ T cells can be pathogenic to the host, but the development of cerebral malaria may require additional factors as has been discussed (10, 11). On the other hand, RAG2-KO OT-I mice showed a clinical course indistinguishable from RAG2-KO mice when infected with WT-PbA, suggesting that CD8+ T cells that are activated in an Ag-nonspecific manner are generally not pathogenic to the host. Nonspecific activation of CD8+ T cells, however, does not require TCR engagement and thus might include a pool of peripheral CD8+ T cells that recognize various MHC class I-bound epitopes including self-Ag. Therefore, it remains possible that activation and CTL development of the self-reactive pool of peripheral CD8+ T cells could lead to the destruction of tissue and might be involved in the pathogenesis of malaria. Further studies on the molecular mechanisms underlying the malaria-specific and nonspecific activation of CD8+ T cells are important for expanding our understanding of protection against Plasmodium infection and of the pathogenesis of severe malaria.


   Acknowledgments
 
We thank Drs. H. Kosaka, W. R. Heath, Y. Takahama, and Y. Yoshikai for providing mice; M. Ueda, T. Ikeda, and K. Kimura for technical assistance; Y. Akiyama for help; and Dr. K. Suzue for discussion.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict 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 Grants-in-Aid from the Ministry of Education, Science, Sports and Culture, Japan; and by the 21c COE program at Nagasaki University. Back

2 Address correspondence and reprint requests to Dr. Katsuyuki Yui, Division of Immunology, Department of Molecular Microbiology and Immunology, Graduate School of Biomedical Sciences, Nagasaki University, 1-12-4, Sakamoto, Nagasaki, 852-8523 Japan. E-mail address: katsu{at}nagasaki-u.ac.jp Back

3 Abbreviations used in this paper: PbA, Plasmodium berghei ANKA; OVA-PbA, recombinant Plasmodium berghei ANKA expressing OVA; WT-PbA, wild-type Plasmodium berghei ANKA; hsp, heat shock protein; DHFR-ts, dihydrofolate reductase-thymidyltransferase-ts; KO, knockout; LCMV, lymphocytic choriomeningitis virus; DC, dendritic cell; CD62L, CD62 ligand; OVAp, OVA257–264 peptide. Back

Received for publication October 19, 2007. Accepted for publication May 13, 2008.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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