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*Malaria
The Journal of Immunology, 2000, 164: 1463-1469.
Copyright © 2000 by The American Association of Immunologists

Liver CD4-CD8- NK1.1+ TCR{alpha}ß Intermediate Cells Increase During Experimental Malaria Infection and Are Able to Exhibit Inhibitory Activity Against the Parasite Liver Stage In Vitro1

Sylviane Pied2,*,{dagger}, Jacques Roland{dagger}, Anne Louise{dagger}, Danièle Voegtle{dagger}, Valérie Soulard*, Dominique Mazier* and Pierre-André Cazenave{dagger}

* Institut National de la Santé et de la Recherche Médicale U313, Immunobiologie Cellulaire et Moléculaire des Infections Parasitaires, CHU Pitié-Salpêtrière, Paris, France; and {dagger} Centre National de la Recherche Scientifique Unité de Recherche Associée 1961, Unité d’Immunochimie Analytique, Département d’Immunologie, Institut Pasteur, Paris, France


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Experimental infection of C57BL/6 mice by Plasmodium yoelii sporozoites induced an increase of CD4-CD8- NK1.1+ TCR{alpha}ßint cells and a down-regulation of CD4+ NK1.1+ TCR{alpha}ßint cells in the liver during the acute phase of the infection. These cells showed an activated CD69+, CD122+, CD44high, and CD62Lhigh surface phenotype. Analysis of the expressed TCRVß segment repertoire revealed that most of the expanded CD4-CD8- (double-negative) T cells presented a skewed TCRVß repertoire and preferentially used Vß2 and Vß7 rather than Vß8. To get an insight into the function of expanded NK1.1+ T cells, experiments were designed in vitro to study their activity against P. yoelii liver stage development. P. yoelii-primed CD3+ NK1.1+ intrahepatic lymphocytes inhibited parasite growth within the hepatocyte. The antiplasmodial effector function of the parasite-induced NK1.1+ liver T cells was almost totally reversed with an anti-CD3 Ab. Moreover, IFN-{gamma} was in part involved in this antiparasite activity. These results suggest that up-regulation of CD4-CD8- NK1.1+ {alpha}ß T cells and down-regulation of CD4+ NK1.1+ TCR{alpha}ßint cells may contribute to the early immune response induced by the Plasmodium during the prime infection.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Cellular immunity is regarded as an important mechanism in resistance to infection against liver stages of malaria parasite (1). T cell-mediated immunity induced by the sporozoite, the first invasive form of the malaria parasite, and by the subsequent liver stage is directed against the infected hepatocytes able to process and present malaria Ags to various effector T cells (2, 3). These cell-mediated immune mechanisms have been extensively studied in murine models in which acquired resistance to the malaria liver stage is contingent upon the activation of CD4+ and CD8+ T cell subsets from the {alpha}ß lineage (4, 5). Effective protection against the preerythrocytic stage largely depends on effector mechanisms induced by Th1 cytokines such as IFN-{gamma} (3, 6, 7), IL-1 (6), IL-6 (8, 9), IL-12 (10, 11), and TNF-{alpha} (12), which are able to inhibit or destroy the intrahepatic parasite via induction of the NO (10, 11, 12, 13, 14, 15), reactive radical oxygen intermediate pathways (8), and production of acute phase proteins (16). {gamma}{delta} T cells also contribute to immunity against preerythrocytic stages (17, 18). Although functional properties of immune T cells in host defense against liver stage development have been extensively studied, the basis for the selection, induction, coordination, and maintenance of the various immunocompetent cells present in the liver during the infection remains to be elucidated.

T lymphocyte subpopulations of the liver contain, in addition to the conventional single-positive CD4+ and CD8+ T cells, a particular set of CD4+CD8- or CD4-CD8- (DN)3 (3) TCR{alpha}ß cells representing 40–50% of the lymphocyte population found in the liver (19, 20). These cells exhibit unusual properties because they express the NK1.1 marker (NK1.1+), lower levels of TCRs (TCRint) than conventional T cells, and a variety of NK cell markers, including CD16, Ly-49A, Ly-49C, and CD122 (ß-chain of the IL-2R) (21, 22, 23). Liver NK1.1+ TCR{alpha}ß cells have a restricted usage of the TCRVß gene, mainly Vß2, Vß7, and Vß8.2, and a single V{alpha} domain V{alpha}14 (24, 25), suggesting that these cells are selected by nonpolymorphic ligands. It has been reported that NK1.1+ TCR{alpha}ß cells may recognize hydrophobic Ags, particularly lipids and glycolipids involving presentation by CD1 molecules (26, 27, 28, 29, 30, 31, 32, 33). It is noteworthy that DN {alpha}ß T cells account for a significant proportion of the T cells in other cellular compartments (34, 35), while they are rare in peripheral lymphoid organs and in the blood (23). This unusual liver T cell subset predominantly produces IL-4 (36, 37, 38, 39) or IFN-{gamma} (40), and it has been suggested that it may develop independently of the thymus (21, 22, 41).

In the present study, we have examined the kinetics of T cell responses induced in the liver of C57BL/6 mice during a malaria infection initiated by the injection of P. yoelii sporozoites. We found that P. yoelii induces the increase of intrahepatic DN NK1.1+ {alpha}ß T cells during the acute phase of the infection and after remission, whereas CD4+ NK1.1+ {alpha}ß T cells were down-regulated.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mice and parasite infection

C57BL/6 mice were purchased from Charles River (St-Aubin les Elbeuf, France). Mice were used at 6–10 wk of age. P. yoelii yoelii 265 BY strain, maintained as described previously (42), was used for these experiments. Sporozoites were obtained from infected salivary glands of Anopheles stephensi mosquitoes, 16 to 21 days after an infective blood meal. After aseptic dissection, salivary glands were homogenized in a glass grinder and diluted in sterile PBS. Mice were infected by i.v. injection of 4000 sporozoites. Control animals were injected with sterile PBS. Parasitemia was monitored by detecting parasites every day in blood smears after Giemsa staining.

Antibodies

mAbs specific for mouse CD3 {epsilon}-chain (145-2C11), CD4 (H129.19), and CD8-{alpha} (53-6.7) were obtained from Boehringer Mannheim (Meylan, France). Biotin-conjugated anti-TCR{alpha}ß (9H57-597), anti-TCR{gamma}{delta} (GL3), anti-NK-1.1 (PK 136), anti-CD69 (H1.2F3), anti-CD44 (IM-7), anti-CD62L (Mel-14), and R-PE anti-CD122 (TM-ß1) were purchased from PharMingen (Clinisciences, Montrouge, France). mAbs to the different TCRVß gene families, Vß2 (B20.6), Vß3 (KJ25), Vß4 (KT4), Vß6 (RR4-7), Vß7 (TR310), Vß8.1,2 (KJ16), Vß8.1,2,3 (F23.1), Vß8.2 (F23.2), Vß9 (MR10-2), Vß10 (B21.5), Vß11 (RR3-15), Vß12 (MR11-1), Vß13 (MR12-3), and Vß14 (14.2), were all biotinylated according to the procedure described by Guesdon et al. (43). R4-6A2, a purified rat IgG1 anti-mouse IFN-{gamma} (hybridoma ATCC HB 170), was a gift of G. Milon (Institut Pasteur, Paris, France).

Cell preparation

Livers were removed from control uninfected mice and mice infected with sporozoites 3, 10, and 30 days after injection. Liver lymphocytes (iHLs) were prepared as described by Watanabe et al. (44). Briefly, the liver was passed through stainless steel mesh and suspended in RPMI medium. After one washing, the cells were resuspended in 30% Percoll containing 100 U/ml heparin and centrifuged at 2600 rpm for 20 min at room temperature. The pellet was resuspended in ACK (ammonium chloride/potassium) buffer to lyse erythrocytes and washed twice in 3% FCS-PBS before counting.

FACS analysis and cell sorting

Staining of iHLs was performed, at 4°C for 30 min, by incubating the cells first with biotinylated mAb described before and subsequently with anti-CD3 FITC, anti-CD4 FITC, or anti-CD8 FITC in the presence of PE-conjugated streptavidin. For three-color analysis, Tri-color-conjugated streptavidin was used as third-color reagent. After washing twice, cell analysis was done with a FACScan cytofluorometer (Becton Dickinson, Grenoble, France) using CellQuest software. Viable lymphocytes were carefully gated by forward and side scatter. Analysis was done for each sample on 10,000 acquired events. The percentage of fluorescent positive cells was determined by integrating profiles determined on the basis of viable lymphocytes.

Enrichment in CD3int NK1.1+ T cells and CD3high NK.1.1- T cells was done by sorting performed with a FACStar (Becton Dickinson). Mononuclear cells from liver, removed 10 days after sporozoite inoculation, were two color stained with FITC-conjugated anti-CD3 and PE-conjugated anti-NK1.1 (PK 136).

Culture of malaria hepatic stages

C57BL/6 hepatocytes were prepared as described (42) with minor modification. Cells were isolated by collagenase perfusion (Boehringer Mannheim, Mannheim, Germany) of liver fragments and were further purified over a 60% Percoll gradient (Pharmacia Biotech, Uppsala, Sweden). Hepatocyte purity and viability were >95%, as assessed by trypan blue dye exclusion. Cells (8 x 104) were cultured in eight-chamber plastic Lab-Tek slides (Nunc, Naperville, IL) in William’s medium (Life Technologies, Edinburgh, Scotland) supplemented with 5% FCS (Life Technologies), 100 U/ml penicillin-100 µg/ml streptomycin solution (Life Technologies), and incubated at 37°C in 3.5% CO2 for 24 h. After removal of medium from the culture chambers, 5 x 104 sporozoites were added in 100 µl of fresh supplemented medium. Three hours later, medium was replaced by fresh complete medium and 45 h later, cultures were stopped by ethanol fixation.

In vitro assay of parasite liver stage elimination by liver cells

This assay was done as follows: 3 h after addition of sporozoites to hepatocyte cultures, the medium was replaced by CD3int NK1.1+ and CD3high NK1.1- T cell preparations (purity >99%) from infected and uninfected control mice were added. Cultures were incubated for 45 h, with a change of 50 µl of medium 24 h after parasite inoculation. Anti-IFN-{gamma} (dilution 1/100)or anti-CD3 (dilution 1/300) was added with or without NK1.1+ T cells to hepatocyte cultures 3 h after sporozoite inoculation, and was maintained constant throughout the experiment by adding Abs in fresh medium during medium change. Schizont numbers were assessed in triplicate cultures by immunofluorescence staining using hyperimmune sera recognizing P. yoelii liver stages. Percent inhibition was calculated by comparing the number of parasites in the experimental cultures with the number in control wells.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Increase of CD4-CD8- TCR{alpha}ßint cells in the liver of mice infected with P. yoelii

Liver lymphocytes were prepared from naive and P. yoelii-infected C57BL/6 mice. The phenotype of liver T cells was first examined by two-color FACS analysis with anti-TCR{alpha}ß, anti-TCR{gamma}{delta}, and anti-CD3 mAbs at different time points after sporozoite inoculation (3, 10, and 30 days). Results obtained from three separate experiments (Table IGo) showed that the inflammatory response induced in the liver by P. yoelii infection may cause a cellular influx to the site of parasite development or a proliferation of in situ lymphoid cells. Accordingly, the total number of iHLs recovered from the livers of infected mice increased in time when compared with noninfected control mice. The most marked expansion (11.5-fold) was observed at day 10 after sporozoite inoculation, corresponding to the peak of parasitemia. On day 30, more than 1 wk after mice had recovered from the infection, the absolute numbers of all lymphocyte populations were still high compared with control. Both TCR {gamma}{delta} and {alpha}ß cell populations expanded, showing a polyclonal proliferation of the iHLs induced by the parasite.


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  		Table I. Phenotype of liver T cells from noninfected C57BL/6 mice and after infection with P. yoelii

 
{alpha}ß T lymphocytes in the liver have been reported to be a heterogeneous population comprising both conventional cells and an unusual subset, so-called TCR intermediate (TCRint), expressing ~2- to 3-fold lower TCR surface density. As {alpha}ß T cells constitute the major lymphocyte population stimulated in the liver by the parasite, we have examined the profile of TCR{alpha}ß expression during the course of infection. Surprisingly, as shown on Fig. 1GoA, P. yoelii infection did not reduce the proportion of CD3+ TCR{alpha}ßint in the liver because the percentages of both TCR{alpha}ßint and TCR{alpha}ßhigh cells were increased on days 3, 10, and 30 of infection when compared with noninfected control mice. To characterize these TCR{alpha}ß, three-color cytofluorometric analysis was conducted using anti-CD4 and anti-CD8 mAbs. Gating was done on {alpha}ß T cells to display the CD4 and CD8 profiles among these cells. Liver {alpha}ß T cells from control mice were largely composed of the CD4 subpopulation of both low and high intensity. After 3 days of infection, P. yoelii sporozoites induced a proliferation of CD4+ T cells of high intensity and a loss of CD4+ T cells of low intensity (data not shown). Then, on days 10 and 30, a decrease in the number of total CD4+ TCR{alpha}ß cells was observed, showing that this cell subpopulation became a minor population in the liver during the acute phase of the infection (Fig. 1GoB). This decrease in CD4+ TCR{alpha}ß cell subsets was accompanied by an increase in CD8+ TCR{alpha}ß cell subpopulations and the appearance of DN TCR{alpha}ß cells of high and intermediate intensity that persisted at day 30 (Fig. 1GoB). However, when the gating was done on total CD3+ iHL, an increase in the number of total CD4+ was observed at days 3 and 10 after infection. This suggests that this increase in total CD4+ T cells is due to an expansion of CD4+ cells belonging to the {gamma}{delta} lineage (Fig. 1GoC).



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  		FIGURE 1. A, FACS analysis of expression by liver CD3+ T cells isolated from control and infected C57BL/6 mice 3, 10, and 30 days after P. yoelii sporozoite inoculation. Black histograms represent the controls and open histogram, cells from infected mice. Gating was done on small lymphocytes. M1 indicate CD3+ TCR{alpha}ßint cells and M2, CD3+ TCR{alpha}ßhigh cells. B, CD4, CD8, and DN T cells representation among TCR{alpha}ß-positive cells. Recovered liver cells were triple stained with biotin-conjugated anti-CD4 mAb revealed with Tri-color, FITC-conjugated anti-CD8 mAb, followed by PE-conjugated anti-TCR{alpha}ß mAb. DN T cells among TCR{alpha}ß-positive cells were estimated after labeling with FITC-conjugated anti-CD4 + anti-CD8 mAbs. Numbers of lymphomyeloid liver cells recovered from five individual mice per group were naive, 7.45 x 105 ± 7.9 x 103; day 3, 14.2 x 105 ± 7 x 103; day 10, 10. 4 x 106 ± 4.6 x 104; and day 30, 3. 2 x 106 ± 0.9 x 104. C, Representative numbers of CD4+, CD8+, and CD4-CD8- cells among CD3 T cells recovered from liver of mice infected with P. yoelii 3, 10, and 30 days after sporozoite inoculation. Data are represented as the mean number of results obtained from five individuals per group. Similar results were obtained in two separate experiments.

 
P. yoelii infection induces an increase in CD4-CD8- NK1.1+ T cells and a down-modulation of CD4+ NK1.1+ T cells in the liver

Because the liver of C57BL/6 mice contains a major subset of CD4-CD8- {alpha}ß T cells expressing the NK1.1 surface marker (19, 20), we have analyzed the kinetics of expression of this T cell subset by estimating the percentage of these cells among the iHLs from the liver of mice infected with P. yoelii. As shown in Fig. 2Go, the percentage of liver CD3+ NK1.1+ T cells increased significantly during the intrahepatocytic as well as the erythrocytic phase of the parasite development (day 10) and was still high after remission (on day 30 after infection). NK1.1+ are a major subset of T cells in the liver, and can be either CD4+ or DN (19, 20). As we previously showed that the percentage of CD4+ {alpha}ß T cells decreased and that of DN {alpha}ß T cells increased in P. yoelii-infected liver, this suggests that most of the expanded CD3+ NK1.1+ T cells are of DN TCR{alpha}ß phenotype. Accordingly, we can conclude that CD4+ NK1.1+ T cells were down-regulated.



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  		FIGURE 2. Effect of P. yoelii infection on NK1.1+ CD3 T cells in C57BL/6 mice. The data are displayed as a dot plot after gating on small lymphocytes. Absolute numbers of NK1.1+ CD3 liver lymphocytes: uninfected (0.17 x 106 ± 6 x 103), day 3 (0.4 x 106 ± 4 x 103), day 10 (1.35 x 106 ± 0.6 x 103), and day 30 (1.38 x 106 ± 0.5 x 103). Representative results from a total of five mice per group.

 
We further analyzed the surface phenotype of DN {alpha}ß T cells, and as observed in Fig. 3Go, infection by P. yoelii up-regulates the expression of activation surface marker CD69 and the IL-2R ß-chain (CD122) by CD4-CD8- {alpha}ß T cells. Moreover, the relative numbers of CD69+ and CD122+ cells were increased with time and reached a maximum at day 30 (Fig. 3Go), indicating a stimulation and an activation by the parasite itself. At day 3 of infection, no difference was observed between infected and control mice in the expression of CD44 and CD62L. DN TCR{alpha}ß cells presented a CD44low and CD62Llow phenotype, which became CD44high, CD62Llow at day 10 and CD44high, CD62high at day 30. This is consistent with the expansion of cells with a memory phenotype, followed by a proliferation and/or a recruitment of both naive and memory cells.



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  		FIGURE 3. FACS analysis of cell surface phenotype of liver lymphocytes from C57BL/6 mice 3, 10, and 30 days after infection with P. yoelii sporozoites and in control mice. Liver lymphocytes were triple stained with biotin-conjugated anti-CD4 + anti-CD8 mAbs revealed with Tri-color, followed by FITC-conjugated anti-CD3 mAb and with either PE-conjugated anti-CD69, anti-CD44, anti-CD62L, or anti-CD122, respectively. Gating was done on CD4-CD8-CD3+ cells. Data are displayed as a histogram plot that represents the percentage of positive cells for each phenotypic surface marker.

 
TCRVß usage of liver CD3int T cells in P. yoelii-infected mice

As it has been shown that liver NK1.1+ TCR{alpha}ß lymphocytes use a preferential set of Vß genes, namely Vß2, Vß7, and Vß8 (45, 46, 47), we have analyzed TCRVß-chain expression by CD3int T cells expanded in the liver during P. yoelii infection. As observed in two separate experiments (Fig. 4Go), CD3int iHL from infected mice expressed much less Vß8 when compared with uninfected C57BL/6 control mice, whereas the frequency of Vß2+ and Vß7+ cells was higher among CD3int T cells from infected liver. Thus, P. yoelii molecules preferentially select CD3int TCR Vß2+ and/or Vß7+ cells rather than CD3int cells bearing the Vß8 chains.



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  		FIGURE 4. TCRVß usage by liver lymphocytes from uninfected and P. yoelii-infected mice recovered on days 3, 10, and 30 after parasite inoculation. The distribution of the indicated TCRVß segment on CD3int T cells was determined by two-color FACS analysis. Bar chart represents the mean percentage ± SD of five mice studied individually.

 
Inhibitory activity of CD3int NK1.1+ {alpha}ß T cells against P. yoelii liver stages

To characterize the activity of CD3int NK1.1+ T cells, the effect of this cell subset on the intrahepatocytic development of the parasite was analyzed in vitro. For this purpose, CD3int NK1.1+ and CD3high NK1.1- T cells were isolated from livers of C57BL/6 mice either noninfected or 10 days after sporozoite inoculation. Sorting was performed with a FACStar after two-color staining with FITC-conjugated anti-CD3 and PE-conjugated anti-NK1.1. Antiplasmodial activity of these different T cell subpopulations was examined by adding these cells at different ratios to primary cultures of hepatocyte, 3 h after P. yoelii sporozoite inoculation (Fig. 5Go). Both CD3int NK1.1+ and CD3high NK1.1- T cells, primed in vivo with the parasite and isolated 10 days later, greatly inhibited parasite development when compared with CD3int NK1.1+ T cells from uninfected mice. Nevertheless, the efficacy of CD3high NK1.1- T cells was lower than that of CD3int NK1.1+ T cells, except at the ratio of 20 T cells to 1 hepatocyte. Experiments were then performed to determine the way in which parasite inhibition occurred. The effect of CD3int NK1.1+ T cells was almost totally reversed by an anti-CD3 Ab that had no effect by itself on parasite growth (Fig. 6Go). As it was reported that liver CD3int NK1.1+ T cells produce high levels of IL-4 (36, 37, 38, 39) and IFN-{gamma} (40), we addressed whether IFN-{gamma} was implicated in the inhibitory activity of liver CD3int NK1.1+ T cells by adding anti-IFN-{gamma} mAb to the syngenic infected liver cells. Data showed that anti-IFN-{gamma} Abs, when added to the cultures at the same time as CD3int NK1.1+ T cells, were able to partially reverse the inhibition mediated by these cells at different ratios, but much less so than did the anti-CD3 Ab. The anti-IFN-{gamma} alone had no inhibitory activity on parasite development (Fig. 6Go).



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  		FIGURE 5. In vitro antiplasmodial activity of parasite-primed NK1.1+ liver T cells against P. yoelii liver stage. NK1.1+ CD3int liver T cells from control uninfected and P. yoelii-infected mice or NK1.1- CD3+ cells from infected mice were layered at different ratios onto syngeneic hepatocyte cultures 3 h after sporozoite inoculation. Cultures were stopped 45 h later. Data are presented as the mean percentage of reduction in parasite numbers from triplicate wells of three experiments. Parasite growth percentage reduction was calculated by counting numbers of schizonts in 48-h cultures in the presence or absence of liver lymphocytes.

 


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  		FIGURE 6. Effect of anti-CD3 and anti-IFN-{gamma} Abs on NK1.1+ liver T cell-mediated inhibition. NK1.1+ liver T cells from P. yoelii-infected mice (1.2 x 106) were added with anti-CD3 or anti-IFN-{gamma} mAbs to P. yoelii-infected hepatocyte cultures 3 h after parasite inoculation. Cultures were stopped 45 h later. Data are represented as the mean number of parasites in experimental and control wells done in triplicate, for two experiments.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this study, we demonstrate the expansion of the unconventional NK {alpha}ß T cells in the liver of C57BL/6 mice during the acute phase of malaria infection induced by the inoculation of sporozoites of a nonlethal P. yoelii strain. This T cell subset exhibited a CD4-CD8- phenotype and presented all of the characteristics previously described for NK T cells, such as expression of CD122 and intermediate level of TCR{alpha}ß and CD3 molecules (48). DN NK1.1+ {alpha}ß T cells were still present in the liver at high frequency after the mice had recovered from the disease. In parallel to the increase of DN NK1.1+ {alpha}ß T cells, a down-regulation of CD4+ NK1.1+ {alpha}ß T populations cells and an inversion in the ratio of CD4/CD8 were observed in the liver of C57BL/6 mice during the infection. It is worthy of note that DN NK1.1+ {alpha}ß T cells were also found expanded in the liver of mice during infection initiated with erythrocytic stages of the same parasite strain (data not shown). A similar observation was also recently made by the group of T. Abo in C57BL/6 and C3H/He inoculated with P. yoelii yoelii 17X, and P. yoelii yoelii 17XNL, another lethal and nonlethal P. yoelii strain, respectively (A. Weerasinghe, H. Sekikawa, H. Watanabe, and T. Abo, unpublished data).

In addition, the progressive increase with time of the relative number of DN {alpha}ß T cells and the level of expression of the activation surface marker CD69 observed in the livers of infected compared with control mice indicated that these cells are directly or indirectly stimulated and activated by plasmodial products providing either from the intrahepatic stage and/or the erythrocytic stage. This increase in DN NK1.1+ {alpha}ß T cells and the disappearance of CD4+ NK1.1+ {alpha}ß T lymphocyte subpopulations identified in the liver of normal C57BL/6 mice were also recently observed after infection by several other pathogens, thereby suggesting a regulatory role for this subpopulation of {alpha}ß T cells in the early antiparasite-host response by promoting Th1 responses in vivo through down-regulation of IL-4 secretion (49, 50, 51, 52). Further analysis also revealed that the DN {alpha}ß T cells stimulated by P. yoelii at different times after infection have the CD44highCD62Llow and CD44highCD62Lhigh phenotype, suggesting a proliferation and/or a recruitment of both naive and primed DN TCR{alpha}ßint, as indicated by the level of expression of CD69 and CD122.

NK {alpha}ß T cells have been described as using a preferential set of Vß genes and predominantly Vß8, which can represent up to 50% of the TCRVß segments expressed (45, 46, 47). To determine the TCRVß repertoire usage by DN TCR{alpha}ßint liver cells selected during P. yoelii infection, we assessed the percentage of cells using the restricted set of Vßs and found that Vß2 and Vß7 were preferentially used rather than Vß8 gene products. These results also indicated that the DN T cell populations induced by P. yoelii molecules were not self-reactive forbidden clones that have been described belonging to the Vß8+ set (41, 45). Moreover, if Vß2 is preferentially expressed at day 10, both Vß2 and Vß7 were used at day 30, suggesting an evolution in the NK T cell response that may depend on the parasite stage specificity of plasmodial molecules that stimulate these T cell clones.

Our results, which indicate the expansion of DN NK1.1+ TCR{alpha}ßint liver cells during P. yoelii infection, raise the question of the key role of this T cell subset in the immune response against malaria parasite. Because DN NK1.1+ {alpha}ß T cells were present at high frequency only in the liver of the infected mice, and none in the thymus, the spleen, or the blood, we addressed whether a control is exerted by NK T cells upon the development of the liver stage. To obtain more evidence, we analyzed the ability of these cells to inhibit in vitro the intrahepatocytic development of P. yoelii. For technical reasons (low number of cells present in the liver of 3 days postinfected), NK T cells from 10-day infected mice were used in the in vitro assay of intrahepatic parasite elimination. The data indicated that, when added at different ratios to primary cultures of hepatocytes 3 h after sporozoites inoculation, P. yoelii-induced NK1.1+ TCR{alpha}ß+ effector liver cells were able to decrease the number of mature schizonts. Addition of anti-CD3 mAb markedly reduced the antiplasmodial activity of P. yoelii-primed NK1.1+ TCR{alpha}ß+ cells, showing that the inhibitory activity exhibited by these cells required Ag presentation by the infected hepatocyte and recognition by the TCRs. This observation of inhibitory activity of NK T cells from 10-day infected mice raised the concern of parasite stage Ag specificity of this T cell subset against the liver stage. We cannot exclude a possible effect of NK T cell clones induced by blood stage parasite molecules on the liver stage. It is interesting to note also that liver NK T cells from blood stage-infected mice can also destroy P. yoelii erythrocytic stage in vitro (A. Weerasinghe, H. Sekikawa, H. Watanabe, and T. Abo, unpublished data).

The mechanism by which NK1.1+ TCR{alpha}ß+ iHL exert their antiparasite effect remains unclear. Nevertheless, consistent with the detection of Fas ligand mRNA in NK1.1+ T cells described by Arase et al. (53) and the high level of perforin observed in the cytoplasm of activated CD3int NK1.1+ cells (54), we can propose that these liver lymphocytes may eliminate parasites within the hepatocyte through a Fas ligand/Fas- or perforin-mediated mechanism. However, the expression of Fas molecules by infected hepatocytes remains to be demonstrated. On the other hand, cytokines are known to have an important role as mediators and effectors in the host response to plasmodial infection (55). So, the partial but significant reduction by the anti-IFN-{gamma} Ab of the inhibition of P. yoelii intrahepatocytic schizogony induced by parasite-primed NK1.1+ TCR{alpha}ß+ liver cells also suggests that IFN-{gamma} may be produced by these iHLs themselves and play a role in their inhibitory activity (34). IFN-{gamma} is known to directly eliminate liver stages in vitro by eliciting NO-dependent mechanisms (16). On the other hand, an increasing number of reports shows that IL-12 is a major factor in the proliferation and activation of NK1.1+ TCRint cells (56, 57, 58). Because rIL-12 was shown to protect 100% of mice or monkeys against infection with P. yoelii and P. cynomologi sporozoites, respectively, and that this protection is associated with the high plasma level of IFN-{gamma} (11, 12), we postulate that early production of IL-12 by Kupffer cells induced by parasite component(s) may lead to the subsequent expansion and activation of DN NK1.1+ {alpha}ß T cells that produce IFN-{gamma} (40) and in parallel, to the down-regulation of CD4+ NK1.1+ {alpha}ß T cells that secrete IL-4 (37, 39). This regulation of the NK1.1+ liver T cell subpopulation during primary infection by malaria parasite would promote a Th1 response, which is associated with a protective response against the parasite liver stage (instead of a Th2-type response).

Several studies have reported that NK1.1+ TCR{alpha}ß+ cells recognize components presented by the CD1 molecules that are abundantly expressed in murine liver (59, 60). Based on these observations, we hypothesize that DN NK1.1+ TCR{alpha}ß+ cells primed by the plasmodial molecules are able to recognize parasite ligands presented in the context of CD1 expressed by the infected hepatocyte. The absence of any detectable expansion of DN NK1.1+ {alpha}ß T cells in P. yoelii-infected ß2m-/- mice (data not shown), which shows a MHC class I control of the induction of this T cell subset, is in full agreement with this hypothesis, which is the subject of ongoing studies. GPI was defined as a ligand of CD1 molecules and could be a natural ligand candidate for CD1-restricted T cells (61). Moreover, data published by Schofield et al., showing a role for CD1-restricted NK1.1+ TCR{alpha}ß+ cells in the regulation of the IgG responses to the glycosylphosphatidylinositol-anchored Ags of Plasmodium falciparum and Plasmodium berghei circumsporozoite proteins, reinforced the hypothesis of an eventual role of CD1-restricted NK T cells in immune response in vivo during malaria infection (62). Based on the hydrophobic nature, the lack of requirement of strict specific residues of the ligand binding site on the CD1 family members, and the relative oligoclonality of the NK TCR{alpha}ß repertoire, it is obvious to consider that the glycolipid-anchored parasite protein from either preerythrocytic or blood stage infection could induce NK T cell clones that may interfere with both liver and blood stage parasites.

In summary, our data provide evidence for a participation of DN NK1.1+ TCR{alpha}ß lymphocytes induced by the preerythrocytic and/or erythrocytic stages of malaria parasite in the immune response taking place in the liver, the first site of parasite development within the host; precisely how they are involved remains to be elucidated.


   Footnotes
 
1 This work was supported by Grant 4API01 from Caisse Nationale d’Assurances Maladies des Travailleurs Salariés (CNAMTS). Back

2 Address correspondence and reprint requests to Dr. Sylviane Pied, Institut National de la Santé et de la Recherche Médicale U313, Immunobiologie Cellulaire et Moléculaire des Infections Parasitaires, CHU Pitié-Salpêtrière, 91 Boulevard de l’Hôpital, 75643 Paris cedex 13, France. E-mail address: Back

3 Abbreviations used in this paper: DN, double negative; iHL, intrahepatic lymphocyte; TCRint, TCR intermediate. Back

Received for publication December 21, 1998. Accepted for publication November 15, 1999.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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