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International Immunology, Vol. 11, No. 9, 1553-1562, September 1999
© 1999 Japanese Society for Immunology

T cell response in malaria pathogenesis: selective increase in T cells carrying the TCR Vß8 during experimental cerebral malaria

Mariama Idrissa Boubou1, Alexis Collette1,2, Danielle Voegtlé2, Dominique Mazier1, Pierre-André Cazenave2 and Sylviane Pied1,2

1 INSERM U511, Immunobiologie Cellulaire et Moléculaire des Infections Parasitaires,CHU Pitié-Salpêtrière, 75643 Paris Cedex 13, France
2 CNRS URA 1961, Unité d'Immunochimie Analytique, Département d'Immunologie, Institut Pasteur,75724 Paris Cedex 15, France

Correspondence to: S. Pied, INSERM U511, CHU Pitié-Salpêtrière, 91 Boulevard de l'Hôpital, 75643 Paris Cedex 13, France


   Abstract
Top
 Abstract
Introduction
 Methods
 Results
 Discussion
 References
 
To characterize the T cells involved in the pathogenesis of cerebral malaria (CM) induced by infection with Plasmodium berghei ANKA clone 1.49L (PbA 1.49L), the occurrence of the disease was assessed in mice lacking T cells of either the {alpha}ß or {gamma}{delta} lineage (TCR{alpha}ß–/– or TCR{gamma}{delta}–/–). TCR{gamma}{delta}–/– mice were susceptible to CM, whereas all TCR{alpha}ß–/– mice were resistant, suggesting that T cells of the {alpha}ß lineage are important in the genesis of CM. The repertoire of TCR Vß segment gene expression was examined by flow cytometry in B10.D2 mice, a strain highly susceptible to CM induced by infection with PbA 1.49L. In these mice, CM was associated with an increase of T cells bearing the Vß8.1, 2 segments in the peripheral blood lymphocytes. Most Vß8.1, 2+ T cells from peripheral blood lymphocytes of the mice that developed CM belonged to the CD8 subset, and exhibited the CD69+, CD44high and CD62Llow phenotype surface markers. The link between the increase in Vß8.1, 2+ T cells and the neuropathological consequences of PbA infection was strengthened by the observation that the occurrence of CM was significantly reduced in mice treated with KJ16 antibodies against the Vß8.1 and Vß8.2 chains, and in mice rendered deficient in Vß8.1+ T cells by a mouse mammary tumor virus superantigen.

Keywords: cerebral malaria, malaria pathogenesis, Plasmodium berghei ANKA, T cell response, TCR Vß gene expression


   Introduction
Top
 Abstract
 Introduction
Methods
 Results
 Discussion
 References
 
Cerebral malaria (CM) is one of the most tragic complications associated with the erythrocytic stage of the Plasmodium falciparum life cycle. Every year, CM contributes to the death of 2.5 million people, mainly children in tropical countries where malaria is endemic (1). Adults who develop CM generally have little or no immunity to malaria infection. The development of CM is multifactorial, and depends on both host and parasitic factors, i.e. the parasite strain, the immune status and the genetic background of the host (2–5). Although the physiopathology of CM has been extensively investigated, no element has so far emerged that enables prediction of the evolution of P. falciparum infection towards CM.

Different factors participate in the neuropathogenesis of malaria. They seem to include abnormally high production of Th1 cell-derived cytokines such as tumor necrosis factor (TNF)-{alpha} and IFN-{gamma} induced by infected erythrocytes (6–12). These cytokines may play an important role in causing certain pathological changes, by up-regulating the expression of cell surface markers like CD36, ICAM-1, VCAM-1 and chondroitin sulfate A, thus leading to the sequestration of infected erythrocytes, leukocytes and monocytes in the cerebral capillaries (13–17). One mechanistic theory proposed to explain the cytoadherence of parasite-infected red blood cells is the expression of P. falciparum erythrocyte membrane protein-1 and up-regulation of neurovascular endothelial adhesion molecules, which may cause the neurovascular lesions which characterize CM (15,18–20).

The exact role played by T cells in the induction of mechanisms leading to the manifestation of CM is at present unknown. Nevertheless, several observations in patients with P. falciparum infection have suggested a link between T cells and the mechanisms of pathogenicity: these observations include an association between HLA-Bw53 and resistance to severe malaria (21), the polyclonal activation of T cells (22–24) and the subsequent immunosuppression (25,26), the absence of severe pathology in children suffering from a thymic abnormality (27), and the pronounced activation of T lymphocytes bearing TCR V{gamma}9 chains in non-immune individuals developing a primary infection (28,29).

Rodent models of CM that only partially reflect the characteristics of the disease in humans have allowed the clarification of certain mechanisms involved in this pathology, such as the direct involvement of T cells in the immunopathological process of CM. More precisely, neurological signs developed by genetically susceptible mice infected with the P. berghei ANKA (PbA) strain were shown to be strictly dependent on the presence of T lymphocytes (27,30–33) and more specifically CD4+ T cells (34). CD8+ T cells were also recently reported to be an essential component of experimental CM pathogenesis (35,36). Although the expression of the pathology can differ in humans and animal models, the association between the pathogenesis of CM and the cytokine levels observed in mice infected with PbA is very similar to that observed in human CM, especially as regards TNF-{alpha} levels (6,7,9,10,12).

In this study, we have analyzed the repertoire of the TCR Vß chains expressed by lymphocytes from PbA-infected mice which did or did not develop CM to identify key T cells involved in the pathological process. We demonstrated that certain T cell populations may contribute to some of the immunopathological manifestations observed in the severe form of CM, either directly or by the over-activation of other cells.


   Methods
Top
 Abstract
 Introduction
 Methods
Results
 Discussion
 References
 
Mice
Different strains of 6- to 8-week-old mice with the following characteristics were used: C57BL/10.D2 /nOla Hsd (B10.D2, H-2d, I-E+, Vß5 and Vß11 deleted) from Harlan Sprague-Dawley (Gannat, France); C57BL/6 N Crl BR (H-2b, I-E–), BALB/c AnN Crl BR (H-2d, I-E+, Vß3, Vß5, Vß11 and Vß12 deleted) and DBA/2 N Crl BR (H-2d, I-E+, Vß3, Vß5, Vß6, Vß7, Vß8.1, Vß9, Vß11 and Vß12 deleted) from Charles River (Saint-Aubin les Elbeuf, France). TCR{gamma}{delta}–/– mice generated on a C57BL/6 genetic background were kindly provided by Dr P. Peirera (Département d'Immunologie, Institut Pasteur, Paris). (129/SvxC57BL/6) TCR{alpha}ß–/– and CD4–/– mice generated on a C57BL/6 genetic background, and (129/SvxC57BL/6) CD8–/– mice were purchased from the Centre de Développement des Techniques Avancées (Orléans, France). Strains 129/Sv (H-2b) and C57BL/6 (H-2b) are both highly susceptible to PbA-induced CM. Congenic BALB.D2 mice (H-2d, I-E+, Mls1a: Vß6, Vß7, Vß8.1 and Vß9 deleted) were maintained at the Institut Pasteur from a breeding pair kindly given by Dr Martine Bruley-Rosset (INSERM U267, Villejuif, France) (37). BALB.SW mice were obtained from BALB/c AnN Crl BR females infected with mouse mammary tumor virus (MMTV) (SW), which is known to delete Vß6, Vß7, Vß8.1 and Vß9 as a consequence of infection by the milk-borne MMTV (SW) (38).

Parasite
Erythrocytic stages of clone 1.49L of PbA, kindly given by Dr D. Walliker (Institute of Genetics, Edinburgh, UK), were maintained in C57BL/6 mice. This clone was selected for its great capacity to induce CM characterized by neurological signs (ataxia, paralysis, deviation of the head and convulsions) between 6 and 8 days post-infection (39). Blood stages of the clone were used as stabilates [107 parasitized red blood cells (pRBC)/ml in Alsever's solution containing 10% glycerol] stocked in liquid nitrogen. CM was induced by i.p. injection of 106 pRBC infected by PbA. Parasitemia was monitored by daily parasite detection on blood smears after Giemsa staining.

Antibodies
Anti-mouse CD3{varepsilon} (145-2C11), anti-CD4 (H129.19) and anti-CD8-{alpha} (53-6.7) FITC-conjugated mAb were obtained from Boehringer Mannheim (Meylan, France). Biotin-conjugated anti-TCR{alpha}ß (9H57-597), anti-TCR{gamma}{delta} (GL3), anti-CD69 (H1.2F3), anti-CD44 (IM-7) and anti-CD62L (MEL-14) mAb, and phycoerythrin (PE)-conjugated streptavidin were purchased from PharMingen (Clinisciences, Montrouge, France). TriColor-conjugated streptavidin was purchased from Caltag (TEBU, Le Peray-en-Yvelines, France). mAb to the different Vß gene families of the TCR, i.e. anti-Vß2 (B20.6) (40), anti-Vß3 (KJ25) (41), anti-Vß4 (KT4) (42), anti-Vß6 (RR4-7) (43), anti-Vß7 (TR310) (44), anti-Vß8.1, 2 (KJ16) (45, 46), anti-Vß8.1, 2, 3 (F23.1) (47), anti-Vß9 (MR10-2) (48), anti-Vß10 (B21.5) (40) and anti-Vß14 (14.2) (49), were all prepared and biotinylated in the laboratory of the Institut Pasteur; biotin-labeled anti-Vß12 (MR11-1) and anti-Vß13 (MR12-3) were purchased from PharMingen.

Depletion experiments by treatment with mAb
Different groups of mice with Vß-specific T cell depletion were constituted by i.p. injection of newborn B10.D2 mice with anti-Vß8.1, anti-Vß2 or anti-Vß14, or a mixture of anti-Vß2, anti-Vß6 and anti-Vß14 mAb. Mice from the different groups received 50 µg of Na2SO4 fraction of each anti-Vß isolated from ascitic fluid raised in nude mice, 24 h, and 4 and 8 days after birth, and then 100 µg weekly until infection at 7 weeks. Before infection, the effectiveness of the depletion was tested by determining the frequency of T cells bearing the Vß segment concerned in the peripheral blood lymphocytes (PBL) using flow cytometry (FCM). Treatment with mAb anti-TCR Vß depressed the level of T cells bearing the specific Vß segment: Vß8.1, 2 (18.31 ± 1.91 to 1.09 ± 0.83%), Vß2 (6.30 ± 0.66 to 1.78 ± 0.94%), Vß6 (7.54 ± 1.53 to 1.56 ± 1.02%) and Vß14 (7.49 ± 0.70 to 0.84 ± 0.07%).

Lymphocyte preparation and staining
PBL, lymph nodes (LN) and spleen were removed from uninfected control mice and infected mice on day 7 after PbA inoculation, when CM manifestations appeared. To lyse erythrocytes, spleen cells were treated twice with ammonium chloride/potassium buffer and washed twice with PBS containing 3% FCS (Gibco/BRL, France). PBL were isolated on Ficoll-Hypaque solution (Pharmacia, France), washed twice with PBS/FCS and counted. For cytofluorometric analysis, lymphocytes were incubated, first with biotinylated mAb directed against the different Vß gene families, anti-TCR{alpha}ß, anti-TCR{gamma}{delta}, anti-CD69, anti-CD44 or anti-CD62L mAb, then with anti-CD3–FITC, anti-CD4–FITC or anti-CD8–FITC in the presence of PE-conjugated streptavidin or TriColor-conjugated streptavidin.

FCM analysis
Cells were analyzed using a FACScan cytofluorometer (Becton Dickinson, Grenoble, France) and CellQuest software. Viable lymphocytes were carefully gated by light scattering (FSC/SSC). For the analysis of all the Vß chain expressed, 5000–10,000 events were acquired and recorded per sample. The percentage of positive fluorescent cells was determined by integrating profiles on the basis of the numbers of viable lymphocytes.

Statistical analysis
Results were compared with Statview 4.5 software using one-way or two-way ANOVA and Fischer's protected least significant difference. For phenotypic surface marker analysis, Student's t-test was used. P < 0.01 was considered significant.


   Results
Top
 Abstract
 Introduction
 Methods
 Results
Discussion
 References
 
Murine models of CM
To define mice exhibiting different degrees of susceptibility to CM, several strains expressing the same MHC haplotype (H-2d) were infected with clone 1.49L of PbA. As shown in Fig. 1Go(A), three strains of mice (B10.D2, BALB/c and DBA/2) were found to exhibit different phenotypes as regards their ability to develop CM after the inoculation of 106 pRBC. From days 6 to 8 after parasite inoculation, 90% of the B10.D2 mice died of CM, whereas only 40% of the BALB/c and none of the DBA/2 mice developed clinical signs of CM, including ataxia, spasms, and hemi- and tetraplegia. Since CM occurred in a very high proportion of the B10.D2 congenic mice, this strain was chosen as a model for studying the T cell response during CM. In most of the B10.D2 mice infected by PbA, the neuropathology of this disease was preceded by a progressive decrease in body temperature to a critical level of 30°C before death. The mean level of parasitemia in B10.D2 mice at death was 15.53 ± 4.1%. In all three strains, the mice which survived CM died of anemia due to hyperparasitemia during the third week of infection (Fig. 1BGo). Histopathological analysis of the brain of mice that died of CM revealed clear cerebral lesions associated with leukocytes and sequestrated pRBC (B. Lucas et al., unpublished results).



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  		Fig. 1. Mortality from CM of different mouse strains. Mice of the B10.D2 (n = 40), BALB/c (n = 25) and DBA/2 (n = 25) strains with the same haplotype (H-2d) were infected i.p. with 106 parasitized erythrocytes by PbA clone 1.49L. Neurological manifestations which included paralysis (mono-, hemi-, para- or tetraplegia), deviation of the head, ataxia and convulsions appeared 6–8 days post-infection. The remaining infected mice died 21 days after parasite inoculation. They had severe anemia and hyperparasitemia, but no neurological signs. Results indicate mean cumulative numbers of deaths from CM (A) and the mean levels of parasitemia (B) from four separate experiments.

 
Role of {gamma}{delta} and {alpha}ß T cells in the pathogenesis of CM
Seven days after parasite inoculation, the distribution of lymphocyte subpopulations was examined by FCM in the PBL, LN and spleen of the B10.D2 mice. As shown in Table 1Go, infection by PbA induced a great proliferation of T cells in the circulating blood. Seven days after inoculation, the total number of CD3+ T cells among the PBL rose 4.9-fold compared to 1.4-fold in the spleen. No significant difference in the number of lymphocytes was found in the LN compartment. Lymphocytes which had proliferated by day 7 after i.p. injection of PbA erythrocytic stages were derived from both the {alpha}ß and {gamma}{delta} T cell lineages.


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  		Table 1. Phenotype of T cells in mice of the B10.D2 strain infected with PbA clone 1.49L
 
To define the respective roles played by {gamma}{delta} and {alpha}ß T cells in the pathogenesis of murine CM, its incidence was calculated in PbA-infected mice displaying deficient expression of either TCR{gamma}{delta} or TCR{alpha}ß. As shown in Fig. 2Go(A), >60% of {gamma}{delta} T cell-deficient mice exhibited all the clinical signs which characterize CM, whereas the TCR{alpha}ß–/– mice which remained susceptible to infection did not develop CM. These mice died of anemia due to hyperparasitemia 2–3 weeks after parasite inoculation. Note that the absence of T cells of the {gamma}{delta} or {alpha}ß lineages did not change the level of parasitemia compared to the level for C57BL/6 infected mice (TCR{gamma}{delta}–/– and TCR{alpha}ß–/– mice are generated on a C57BL/6 genetic background) as observed in two independent experiments (Fig. 2BGo). We conclude that the absence of T cells from the {gamma}{delta} lineage has no effect on the pathogenesis of CM, whereas {alpha}ß T cells play a critically important role in the genesis and mediation of the immunological mechanisms leading to the development of the neuropathology.



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  		Fig. 2. Role of the {gamma}{delta} and {alpha}ß T cell lineages in the pathogenesis of CM. TCR{gamma}{delta}–/– (n = 20), TCR{alpha}ß–/– (n = 10) and C57BL/6 (n = 17) mice were infected with 106 parasitized erythrocytes by PbA clone 1.49L. Results indicate the mean cumulative numbers of deaths from CM (A) and the mean levels of parasitemia (B) from two separate experiments.

 
TCR Vß repertoire of CD3+ T cells in mice infected with PbA
Using FCM, we examined the expression of the TCR Vß gene repertoire in PBL, LN and spleen cells in B10.D2 mice, 7 days after infection with PbA 1.49L. The percentage of CD3+ T cells bearing the Vß8 segment rose in the PBL of infected mice compared to non-infected controls (Fig. 3AGo). The TCR Vß segments encoded by Vß8.1 and/or Vß8.2, but not by Vß8.3, appeared to be selected during the infection, as shown by the large percentage of cells labeled by the mAb KJ16 that recognizes Vß8.1 and Vß8.2 T cells, and by the F23.1 antibody that recognizes the three members of the Vß8 family. Statistical analysis was done using two-way ANOVA considering organs and infection as criteria. As several TCR Vß segments were analyzed, P < 0.005 was defined to be significant. Results presented in Fig. 3Go(B) showed a strong interaction between the two criteria (P = 0.0015; F280 = 7.077) due to the increase of TCR Vß8.1, 2 in PBL. In this compartment, no significant changes were noted in the frequency of cells expressing other Vß segments in the group of infected mice.



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  		Fig. 3. (A) TCR Vß gene segments expression in PbA-infected B10.D2 mice. Bar charts represent the mean percentages ± SD for control (n = 5) and infected mice (n = 15) at day 7 post-infection from five separate experiments. Mice were studied individually. (B) Interaction diagram obtained from two-way ANOVA by considering organs and infection as criteria. White bars, control mice; black bars, infected mice.

 
Comparison of the frequencies of Vß8.1+ and Vß8.2+ T cells in the blood of uninfected controls, infected mice that developed CM (CM+) and infected mice that did not develop it (CM–) was done using one-way ANOVA (considering criteria of CM and P < 0.01 as significant). Data showed a difference in the frequency of Vß8.1, 2+ T cells between these three groups of mice (P = 0.0018; F226 = 8.109). As shown in Fig. 4Go, this difference was due to an increase in the percentage of cells bearing TCR Vß8.1, 2 chain only in the group of mice manifesting CM [control versus CM– (P = 0.690), control versus CM+ (P = 0.001), CM– versus CM+ (P = 0.0069)]. These data indicate that at day 7 post-infection, a specific T cell subpopulation bearing the Vß8.1+ and/or Vß8.2+ TCR increased in the PBL of the B10.D2 mice that developed CM, whereas no change was observed in the PBL of the DBA/2 mice, none of which developed CM (in control mice Vß8.1, 2, 3+ CD3+ T cells = 24.36 ± 0.07% versus 23.58 ± 2.42% in infected mice).



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  		Fig. 4. Percentages of TCR Vß8.1, 2 chains among CD3+ T cells in PBL of PbA-infected B10.D2 mice. Controls (n = 10) are uninfected mice. CM– (n = 7) and CM+ (n = 12) are PbA-infected mice without and with CM respectively, 7 days after infection. Results indicate the percentages of Vß8.1, 2+ CD3+ T cells in the PBL from two separate experiments. Each mouse is plotted separately. Mean absolute numbers of Vß8.1, 2+ CD3+ T cells at day 7 post-infection: controls = 9.3x104; CM– = 49.1x104 and CM+ = 76.9x104.

 
Characteristics of the Increased of Vß8.1, 2+ T Cells
The distribution of cells bearing the TCR Vß8.1, 2 segments among CD4+ and CD8+ lymphocyte subsets was investigated in PBL by FCM, and results were analyzed by one-way ANOVA with P < 0.01 considered as significant (Fig. 5Go). By comparing control, CM– and CM+ groups of mice, a significant difference in frequency of Vß8.1, 2+ cells was observed in both CD4 (P = 0.0013; F217 = 10.123) and CD8 (P = 0.0019; F218 = 9.048) T cell subsets. When comparison was done between CM– and CM+ mice the increase was significant only in the CD8 subset (P = 0.003). This result led us to estimate the respective requirements of CD4+ and CD8+ T cells during CM. Using CD4–/– and CD8–/– C57BL/6 mice, we found that although both groups of mice remained susceptible to infection by clone 1.49L of PbA, they never developed CM (Table 2Go). This suggests that in our model of CM, both the CD4+ and CD8+ T cell subsets are involved in the immunological mechanisms leading to the development of this pathology.



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  		Fig. 5. Percentages of TCR Vß8.1, 2 chains among CD4+ and CD8+ T cells subsets in PBL from PbA-infected B10.D2 mice. Control (n = 5) are uninfected mice. CM– (n = 4) and CM+ (n = 12) are PbA-infected mice without and with CM respectively. Results are expressed as the percentage of Vß-positive cells in each experimental group after FACS analysis. Each square corresponds to one mouse in two separate experiments. Mean absolute numbers of Vß8.1, 2+ CD4+ T cells at day 7 post-infection: controls = 0.9x104; CM– = 5.7x104 and CM+ = 7.0x104. Mean absolute numbers of Vß8.1, 2+ CD8+ T cells at day 7 post-infection: controls = 0.9x104, CM– = 5.7x104 and CM+ = 9.1x104.

 

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  		Table 2. Role of T cells in the pathogenesis of PbA-induced CM
 
To determine whether infection with PbA resulted in Vß8+-activated PBL T cells, the expression of the CD69 marker on these cells was estimated in vivo by cytofluorometry (Fig. 6Go) Most of these Vß8.1, 2+ T cells were activated as shown by the significant expression of the CD69 marker in CM+ mice compared to non-infected control (P < 0.0001; t = –6.350; d.f. = 11). Furthermore, the expression level of CD44 and CD62L was assessed by FCM. As shown by Student's t-test, the frequencies of Vß8.1, 2+ CD44+ (P < 0.0001; t = –6.181; d.f. = 11) and Vß8.1, 2+ CD62L+ (P < 0.0001; t = –6.445; d.f. = 11) were significantly increased in CM+ mice compared to control (Fig. 6Go). Most of Vß8.1, 2+ T cells from PBL of CM+ mice displayed a CD44high (P < 0.0002; t = –5.333; d.f. = 11) and CD62Llow (P < 0.0002 ; t = –5.474; d.f. = 11) surface phenotype suggesting the activation and proliferation of memory T cells.



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  		Fig. 6. FACS analysis of the expression of CD69, CD44 and CD62L markers on TCR Vß8+ cells in PBL. PBL from B10.D2 mice 7 days after infection with PbA 1.49L were stained (i) with biotinylated anti-Vß8.1, 2 (KJ16) mAb, (ii) with anti-CD3–FITC (FL1) in the presence of streptavidin-conjugated-PE (FL2) and (iii) with anti-CD69, anti-CD44 or anti-CD62L mAb respectively in the presence of streptavidin-conjugated TriColor (FL3). Gating was done on CD3+ T cells. The mean percentages of Vß8.1, 2+ CD69+, Vß8.1, 2+ CD44+ and Vß8.1, 2+ CD62L+ T cells are indicated in the corresponding upper right quadrant. Plots show 100% of gated events.

 
Effect of Vß8+ T cell deletion on the incidence of CM
Several experiments were designed to ascertain whether Vß8.1, 2+ T cells interfere with PbA-induced immunopathology. The first set was performed in B10.D2 mice neonatally depleted by in vivo injection of antibodies specific for different Vß segments. Only 40% of the mice treated with anti-Vß8 antibody (KJ16) died of CM. By contrast, 90–100% of a control group of uninfected B10.D2 mice, which were either untreated or treated with anti-Vß14 antibody alone, or with a mixture of anti-Vß2, anti-Vß6 and anti-Vß14 antibodies (this control group was introduced in order to delete a percentage of cells similar to the percentages of Vß8.1+ and Vß8.2+ cells deleted in the infected B10.D2 mice) developed CM. It is worth noting that in the group of KJ16-treated B10.D2 mice which developed CM, both the appearance of clinical signs and death were delayed (Fig. 7Go). Treatment with antibodies directed against the different Vß segments, whatever the Vß specificity of these antibodies, had no effect on the course of parasitemia (data not shown).



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  		Fig. 7. Effect on CM of in vivo treatment with anti-Vß8 mAb. B10.D2 mice were depleted of TCR Vß8-bearing cells during neonatal life by i.p. administration of the following antibodies: anti-Vß8.1, 2 (n = 17); anti-Vß14 (n = 5) and anti-Vß2 + Vß6 + Vß14 (n = 14). Controls (n = 20) and depleted mice were inoculated i.p. with 106 erythrocytes parasitized by PbA clone 1.49L. Data shown the mean percentages of cumulative mortality from CM in three separate experiments.

 
Another set of experiments was performed in BALB.D2 mice congenic for Mls1a which constitutively deletes Vß6, Vß7, Vß8.1 and Vß9 T cells. After parasite inoculation, BALB.D2 Mls1a mice remained susceptible to infection, but did not develop CM (Fig. 8Go). Only one mouse in the BALB.D2 Mls1a group died of CM on day 11 after inoculation. This result might be due to the fact that the Vß8.1+ T cell population in this mouse was not totally deleted in the PBL. To avoid the possible effect of the genetic background in generating the disease, an additional experiment was done using (BALB.D2 Mls1axB10.D2)F1 mice. As shown in Fig. 8Go, only 20% of these mice developed CM compared to 80% in the control group (BALB/cxB10.D2)F1. To exclude possible interference between MMTV genomic integration and malaria infection, the behavior of BALB.SW mice was studied. These mice, bearing the exogenous MMTV (SW) which induces peripheral deletion of the same set of T cells as those deleted in BALB.D2 Mls1a mice, were found to be as resistant to CM as the latter mice.



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  		Fig. 8. Occurrence of CM in mice lacking Vß8.1+ T cells. The following strains of mice were inoculated i.p. with 106 erythrocytes parasitized by PbA clone 1.49L: B10.D2 (n = 40), BALB. D2 Mls1a (n = 20), BALB/c (n = 25), BALB.SW (n = 10), (BALB/cxB10.D2)F1 (n = 34) and (BALB.D2 Mls1axB10.D2)F1 (n = 16). The mean percentage of cumulative mortality from CM in each strain is plotted from four separate experiments.

 

   Discussion
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
References
 
The present results indicate that, 7 days after infection by erythrocytic stages of PbA, the number of T cells of both the {alpha}ß and {gamma}{delta} lineages increased in the blood and spleen of B10.D2 mice.

To assess the respective parts played by {gamma}{delta} and {alpha}ß T cells in the immunopathological process, the incidence of CM was studied in mice deficient in either {gamma}{delta} or {alpha}ß T cells, after their infection with PbA. The mice deficient in {gamma}{delta} T cells were found to be susceptible to CM, whereas those deficient in {alpha}ß T cells were protected against it. The different behavior observed in the two groups of mice was not due to their direct involvement in parasitic development, since the degree of parasitemia in both groups was similar. The failure of TCR{alpha}ß-deficient mice to develop CM indicates that {gamma}{delta} T cells are not necessary for the pathogenesis in the experimental murine CM induced by PbA. These results are not compatible with the hypothesis that the extensive recruitment of {gamma}{delta} T cells in PBL induced by the malaria parasite helps to determine the severity of CM through the production by these cells of TNF-{alpha} and IFN-{gamma} (28,29,50,51), both of which are known to be important mediators in the pathogenesis of CM (6,7,10,12,52). The increase in the number of {gamma}{delta} T cells that we observed during primary infection with PbA erythrocytic stages is in agreement with previously published reports relating the in vivo and in vitro increases in {gamma}{delta} T cells from spleen or blood to a non-lethal infection by Trypanosoma cruzi (53) and several species of murine Plasmodium (54–57).

Our results demonstrate the importance of T cells of the {alpha}ß lineage in the immunopathogenesis of murine CM. To determine whether the immunopathological reaction associated with CM development depends on the presence of specific T cell subpopulations, TCR Vß gene repertoire expression was determined ex vivo in B10.D2 mice with or without the clinical symptoms of CM. Five sets of conclusions were drawn from the data thus obtained. (i) A restricted subpopulation of cells bearing a specific TCR Vß segment may be involved in the immunopathogenesis of CM, because cerebral complications in susceptible H-2d mice were found to be associated with a rapid increase in the number of T cells bearing a receptor encoded by the Vß8 gene family for a short period just before death, which occurred 6–8 days after parasite inoculation. (ii) The fact that the preferential increase in the number of Vß8.1, 2+ T cells was only observed in PBL and not in LN or spleen suggests that the response is very compartmentalized. (iii) The increase in Vß8.1+ and/or 8.2+ T cells only appeared in mice manifesting severe neurological signs, and not in infected mice which did not developed CM. Furthermore, the frequency of Vß8.1, 2+ T cells was well correlated with the severity of the disease. The involvement of these specific cells in murine CM pathogenesis was strengthened by the observation that mice of the DBA/2 strain, another H-2d strain which is not susceptible to CM, did not exhibit an increased number of T cells bearing the TCR Vß8 segment when infected with PbA. (iv) The increase in the number of cells bearing this TCR Vß segment occurred in both the CD4+ and CD8+ PBL subsets, suggesting that these subpopulations were both directly or indirectly involved in the cell-mediated pathogenesis. (v) Assessment of phenotypic marker expression showed that the increased subpopulation of Vß8.1, 2+-bearing cells in the PBL of B10.D2 mice that developed CM exhibited up-regulation of the early activation marker CD69, and expressed the phenotype of memory T cell markers CD44high and CD62Llow.

Whereas it was previously reported that only CD4+ T cells have an important role in the pathogenesis of CM (34), our findings show that the numbers of both the CD4 and CD8 T cells bearing the appropriate TCR Vß8 chain increased, and that, surprisingly, many more Vß8+ CD8+ T cells were present in the mice that developed CM than in those that did not. These data indicate that CD8+ T cells have a role in the pathogenesis of CM which is still unclear. As regards the potential role of the CD8+ T cell subset, treatment with anti-CD8 was recently reported to have prevented the development of experimental CM in C57BL/6 mice infected with PbA, even when it was performed during end-stage disease, shortly before death was expected (36). Other reports stated that ß2-microglobulin-deficient (ß2m–/– mice), which are deficient in CD8+ T cells, are also more resistant to CM than normal control mice (35). These observations disagree with those made by Grau et al. (34), who stated that CD4+ T cells, but not CD8+ T cells had a role in the pathogenesis of experimental CM. Here, the use of CD8–/– mice enabled us to provide direct evidence that CD8+ T cells act as effector cells in the pathogenesis of CM. CD8–/– and ß2m–/– mice differ, in as much as ß2m–/– mice still possess some functional CD8+ T cells (58,59). Mice with a defective expression of ß2m failed to express MHC class I molecules, which were shown to be implicated in susceptibility to severe malaria in humans (60). The absence of CM in ß2m–/– mice might be due as much to the lack of MHC class I molecules as to the deficiency of CD8+ T cells. Mice that lack CD8+ T cells possess CD4+ T cells which are fully functional (61). Mice lacking CD4+ T cells exhibit normal development and normal CD8+ T cell functions, and, in addition, a small subpopulation of TCR{alpha}ß CD4–CD8– cells (62). The present observations that (i) the numbers of T cells bearing TCR from the Vß8 family increased among both the CD4+ and CD8+ T cell subsets during CM, and (ii) CD4–/– and CD8–/– mice were resistant to CM when infected with PbA constitute a direct demonstration that both CD4+ and CD8+ T cells are essential for the development of CM. On the other hand, CD4 or CD8 separately were not sufficient to induce CM, suggesting either cooperation between CD4 and CD8 T cells or the existence of a regulatory circuit including both subpopulations.

The fact that the increase in Vß8.1, 2+ T cells was only seen in PBL suggests the occurrence of either a local proliferative response or a selective recruitment of these cells in the circulation. One possible explanation for the sudden huge expansion of this subpopulation bearing the TCR Vß8 chain just before death is the existence of superantigenic activity during infection by PbA, as demonstrated for P. yoelii infection (63). Superantigens are molecules derived from a number of pathogens that stimulate T lymphocytes subpopulations through a particular TCR Vß chain (64). In general, T cell activation by superantigens is followed by the depletion or inactivation of cells bearing the appropriate Vß chain. Nevertheless, superantigenic activity cannot be responsible for the selective enrichment in Vß8+ T cells observed here in the PBL of dying mice, because it was not detected in other anatomical compartments including the spleen and LN, or in mice neonatally infected with PbA pRBC (data not shown). In addition, it was repeatedly observed that all individuals of the same inbred mice strain displayed the same behavior after a given superantigenic stimulation. Thus, the mice that did not develop CM but were infected with the same batch of parasites as those that did exhibited no increase in the numbers of CD4 or CD8 cells bearing the TCR Vß8 segment. An alternative explanation for the preferential increase in these T cells in the mice developing CM may be a clonal restricted response against one or several dominant antigenic epitopes, as reported in Leishmania major infection (65).

The link between the increase in T cells bearing the Vß8.1, 2 segment and the pathological consequences of PbA infection is supported by our observation that the occurrence of CM was significantly reduced in mice treated with the KJ16 antibody against Vß8.1 and Vß8.2. Similarly, BALB.D2 congenic mice for Mls1a which deletes Vß8.1+ T cells, as well as (BALB.D2 Mls1axB10.D2)F1, BALB.SW and B10.D2 mice nursed by BALB/c mothers infected with MMTV (SW) which induces progressive deletion of Vß8.1 T cells (data not shown), did not develop CM when infected with asexual blood stages of PbA. These data strongly suggest that in B10.D2 mice, highly restricted subsets of Vß8.1+ CD4+ and Vß8.1+ CD8+ T cells are involved in the pathogenic mechanisms of CM.

As already stated, the mechanism by which these T cells mediate CM is still unclear. Since experimental CM has been characterized as a Th1 cell-derived cytokine-dependent disease, it is conceivable that Vß8+ CD4+ T cells are involved in the pathogenesis of CM through the pattern of cytokines they produce. CD8+ T cells can also be classified into types 1 and 2, according to their specific phenotype and cytokine production (66,67). It is therefore possible that CD8+ T cells help to cause neurovascular damage, either indirectly by exerting cytokine-mediated effects or via their cytotoxicity. Consequently, the possibility that in CM, the expanded Vß8+ CD8+ cell populations act as potent regulators of pathogenic T cells should not be excluded and we are currently testing this hypothesis.


   Acknowledgments
 
We thank Drs Charles Elson, Gordon Langsley and Adrien Six for helpful discussion and advice, Mr Maurel Tefit for animal care, Mrs Marie-Christine Wagner for FCM analysis, Mrs Monique Bauzou and Sylvie Euvrard for parasite detection on blood smears, Mr Olivier Gorgette for statistical analysis, and Mrs Michèle Berson for help in preparing the manuscript. This work was supported by a grant from CNAMTS (no. 4API01). M. I. B. is the recipient of a fellowship awarded by the Ministère de l'Education Nationale, de la Recherche et de la Technologie.


   Abbreviations
 
ß2mß2-microglobulin
CMcerebral malaria
FCMflow cytometry
LNlymph nodes
MMTVmouse mammary tumor virus
PbAPlasmodium berghei ANKA
PBLperipheral blood lymphocyte
PEphycoerythrin
pRBCparasitized red blood cell
TNFtumor necrosis factor

   Notes
 
Transmitting editor: S. H. E. Kaufmann

Received 8 December 1998, accepted 1 June 1999.


   References
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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S. Bagot, F. Nogueira, A. Collette, V. do Rosario, F. Lemonier, P.-A. Cazenave, and S. Pied
Comparative Study of Brain CD8+ T Cells Induced by Sporozoites and Those Induced by Blood-Stage Plasmodium berghei ANKA Involved in the Development of Cerebral Malaria
Infect. Immun., May 1, 2004; 72(5): 2817 - 2826.
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E. Belnoue, M. Kayibanda, J.-C. Deschemin, M. Viguier, M. Mack, W. A. Kuziel, and L. Renia
CCR5 deficiency decreases susceptibility to experimental cerebral malaria
Blood, June 1, 2003; 101(11): 4253 - 4259.
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J. Nitcheu, O. Bonduelle, C. Combadiere, M. Tefit, D. Seilhean, D. Mazier, and B. Combadiere
Perforin-Dependent Brain-Infiltrating Cytotoxic CD8+ T Lymphocytes Mediate Experimental Cerebral Malaria Pathogenesis
J. Immunol., February 15, 2003; 170(4): 2221 - 2228.
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E. Belnoue, M. Kayibanda, A. M. Vigario, J.-C. Deschemin, N. v. Rooijen, M. Viguier, G. Snounou, and L. Renia
On the Pathogenic Role of Brain-Sequestered {alpha}{beta} CD8+ T Cells in Experimental Cerebral Malaria
J. Immunol., December 1, 2002; 169(11): 6369 - 6375.
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O. Gorgette, A. Existe, M. I. Boubou, S. Bagot, J.-L. Guenet, D. Mazier, P.-A. Cazenave, and S. Pied
Deletion of T Cells Bearing the V{beta}8.1 T-Cell Receptor following Mouse Mammary Tumor Virus 7 Integration Confers Resistance to Murine Cerebral Malaria
Infect. Immun., July 1, 2002; 70(7): 3701 - 3706.
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S. Bagot, M. Idrissa Boubou, S. Campino, C. Behrschmidt, O. Gorgette, J.-L. Guenet, C. Penha-Goncalves, D. Mazier, S. Pied, and P.-A. Cazenave
Susceptibility to Experimental Cerebral Malaria Induced by Plasmodium berghei ANKA in Inbred Mouse Strains Recently Derived from Wild Stock
Infect. Immun., April 1, 2002; 70(4): 2049 - 2056.
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J. Wipasa, H. Xu, A. Stowers, and M. F. Good
Apoptotic Deletion of Th Cells Specific for the 19-kDa Carboxyl-Terminal Fragment of Merozoite Surface Protein 1 During Malaria Infection
J. Immunol., October 1, 2001; 167(7): 3903 - 3909.
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