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The mechanisms whereby cells and molecules of the immune system function in immunity to malaria and the pathogenesis of this disease are ill-defined. Ho et al. (6) and others (13) have reported that humans with Plasmodium falciparum malaria exhibit a marked increase in the number of peripheral blood T cells, which remain elevated for more than a month after cure. This observation led us to suggest that T cells may exert a protective function during malaria. Subsequently we reported that cloned T cells are cytotoxic for P. falciparum in vitro (3) and that T cells are essential for the expression of cell-mediated immunity in vivo against the murine malarial parasite P. chabaudi (16).

Others have suggested that T cells cause certain pathologic changes associated with malaria (10). Roussilhon et al. (14), for example, observed that human T-cell clones in long-term cultures proliferate and exert cytotoxic activity in response to stimulation with autologous T-cell clones. These authors contend that regulatory interactions occur between activated T cells and T cells during malaria and may lead to temporary immunodepression of T-cell responses and the initial lymphocytopenia associated with infection. In addition, Perera et al. (11) reported that the severity of gastrointestinal symptoms in patients infected with P. vivax correlates with the number of T cells in peripheral blood.

Although the exact role T cells play in cerebral malaria (CM) has not yet been established, it is possible that these cells, which produce an array of cytokines, including gamma interferon (IFN-) and tumor necrosis factor alpha (9), may function in the pathogenesis of this disease. To address this possibility, we have examined the ability of P. berghei ANKA to produce CM in mice depleted of T cells by antibody treatment or gene knockout (KO). We report here that the depletion of T cells during adult life protects mice against CM; in contrast, mice genetically deprived of these cells throughout life develop CM when infected with P. berghei.

Pathogen-free male and female C57BL/6 mice (H-2^b) were purchased from the Jackson Laboratory (Bar Harbor, Maine). Pathogen-free ^0/0 mice, which lack the  chain of the T-cell receptor (TCR) and thus lack detectable T cells (7), were originally purchased from the same source and then were bred and maintained at the University of Wisconsin Gnotobiotic Laboratory (Madison, Wis.). These mice had a mixed 129/C57BL/6 (H-2^b) genetic background and were predominantly of the C57BL/6 phenotype due to their four backcrosses to the C57BL/6 strain. Early in the study, we determined experimentally that both parental strains are highly susceptible to induction of CM by P. berghei ANKA, with 70 to 100% of mice infected with a dose of 10⁶ parasitized erythrocytes manifesting this disease (unpublished data). Moreover, Yañez et al. reported earlier (17) that phenotypically normal heterozygote littermates to three other KO variants on the same 129/C57BL/6 background were also in this same susceptibility range (averaging 71% development of CM). We therefore routinely used C57BL/6 mice as controls in our experiments, because our breeding protocol for this particular KO (^0/0 × ^0/0) did not generate heterozygous littermates. All mice used were between 6 and 8 weeks of age.

Infections with P. berghei parasites were initiated by intraperitoneal (i.p.) injection of blood containing 10⁶ parasitized erythrocytes from a parasitized ^0/0 donor, as described previously (17). We chose this standardized inoculum because we have consistently observed that within the range of 10⁵ to 10⁷ parasitized erythrocytes, a few susceptible mice (10 to 30%) may not develop CM in any given group. Mice were sacrificed when they became moribund. Spleens were removed and prepared for flow cytometric analysis; brains were fixed in 10% neutral buffered formalin for histological examination. Mice were judged to have CM only if they displayed neurological signs (ataxia, seizures, and/or paralysis), became moribund within the first 2 weeks of infection (6 to 14 days postinoculation [p.i.]), and exhibited neurological lesions (hemorrhage, mononuclear cell accumulation within cerebral vessels, edema, and/or endothelial damage) upon histological examination of fixed, hematoxylin and eosin-stained sections of brain tissue (17). Parasitemia was assessed from Giemsa-stained thin smears of tail blood prepared every 3 to 4 days p.i.; the percentage of parasitized erythrocytes was determined by counting between 200 and 1,000 erythrocytes.

T cells were depleted in vivo by treatment with TCR -specific (hybridoma clone GL3) monoclonal antibody (MAb). High-performance liquid chromatography-purified hamster anti-TCR MAb was injected i.p. into each C57BL/6 mouse (six mice per group) at a dose of 0.5 mg on days 0 and 4 p.i. A purified hamster immunoglobulin G (IgG) (Accurate Chemical & Scientific, Westbury, N.Y.) was injected identically into an equal number of C57BL/6 controls. The efficacy of T-cell depletion in infected mice was determined by two-color flow cytometry of spleen lymphocytes, as described previously (4). On day 6 p.i. T-cell-depleted P. berghei-infected C57BL/6 mice showed <0.01% splenic T cells compared to 0.60% in hamster Ig-treated controls. Mice (three per group) were also depleted of T cells with a single dose (0.8 mg) of anti-TCR injected at different times during the course of infection. Control mice were injected with the same dose of hamster Ig. Other mice (three per group) were depleted of CD4⁺ or CD8⁺ T cells with a CD4-specific (hybridoma clone GK 1.5) or CD8-specific (hybridoma clone 2.43) MAb with a dose of 0.7 mg/mouse. Rat Ig (0.7 mg/mouse; Accurate Chemical & Scientific), was injected i.p. into control mice. All monoclonal antibodies were kindly provided by C. Czuprynski (University of Wisconsin, Madison).

The pattern of death in mice infected with P. berghei is known to be biphasic: mice either die within the first 2 weeks of infection with CM, or they die after 3 to 4 weeks of infection with severe anemia and hyperparasitemia, but no neurological manifestations (1). We observed that nearly all infected mice treated with hamster Ig became moribund with CM by day 7 p.i. In contrast, none of the mice depleted of T cells developed CM (Table 1), but instead they became moribund without pathological signs of CM after day 21 p.i. Moreover, histological signs of CM were never observed in the brains of mice that had been depleted of T cells, whether sacrificed on day 7 p.i. or on the day of moribundity after day 21 p.i., when these mice were severely anemic and hyperparasitiemic.

View this table: [in this window] [in a new window]   TABLE 1.   Effects of T-cell depletion on the development of CM in mice infected with P. berghei ANKA

Mice were next depleted of T cells at different times during the course of P. berghei infection with a single dose of MAb to determine the time at which these cells participate in the development of CM. For comparative purposes, other mice were depleted of CD4⁺ or CD8⁺ T cells; both T-cell subsets are crucial for the pathogenesis of P. berghei-induced CM (17). The results in Table 2 suggest that T cells as well as CD4⁺ or CD8⁺ T cells function prior to day 5 of infection. A second mouse model deficient in T cells, the ^0/0 mouse, was examined for its ability to resist development of CM when infected with P. berghei ANKA. Although none of the adult mice depleted of T cells by antibody treatment before day 5 of infection ever showed any physical or histologic evidence of CM, a substantial number (12 of 28 [54%]) of the T-cell-deficient KO mice developed CM that was easily recognizable by physical signs and clearly verifiable histologically.

View this table: [in this window] [in a new window]   TABLE 2.   Development of CM in mice depleted of T cells at different times during the course of P. berghei malaria

Our current findings indicate that T cells, in addition to CD4⁺ and CD8⁺ T cells, have an essential function in the pathogenesis of CM. Although the relationship between these T-cell subsets remains to be determined, the results of our depletion studies at different times during infection suggest that they all function within several days after the initiation of infection. Unfortunately, the depletion of T-cell subsets at the times we chose does not permit us to determine whether these subsets function in a synergistic fashion or independently. We have observed that the activation of T cells during experimental P. chabaudi malaria is dependent upon the presence of CD4⁺ T cells (15). CD4⁺ T cells activated by malarial antigens appear to produce cytokines that activate T cells to proliferate and function in protective cell-mediated immunity against this parasite (2, 16). In contrast, others have suggested that T cells function as components of the innate immune system (4, 8). When stimulated by pathogens, these cells act as a first line of defense and are thought to function by cytokine activation of cells of the adaptive immune response in a manner analogous to activation by NK cells. T cells produce IFN- and tumor necrosis factor alpha in response to stimulation with different antigens (9) and appear to be the major source of IFN- when human peripheral blood mononuclear cells are cultured in the presence of P. falciparum antigens (12). Our observation that T cells appear to function early during infection at a time when little if any proliferation of the T-cell subset has occurred suggests that they may act in a similar capacity, i.e., as helper or stimulating cells rather than as effector cells in the pathogenesis of CM. Although the mechanisms remain to be determined, IFN- appears to be essential for the development of CM (5, 12, 17); it is possible that T cells activated by malarial antigens early during infection secrete IFN-, which initiates events leading to disease.

The results of our studies with ^0/0 mice were unexpected (Table 3). About half of the T-cell-deficient mice in each of two experiments developed CM when infected with P. berghei ANKA. These mice, which lack T cells throughout life, may develop compensatory cells that eventually contribute to the pathogenesis of CM. It seems possible that under certain circumstances, different T-cell subsets or combinations of subsets may function independently of each other to produce disease. Whether T cells have an essential function in the pathogenesis of human CM remains to be determined. However, should they have such a function, the deletion of T cells or the neutralization of crucial cytokines secreted by T cells could provide a new approach to the therapy of human CM, particularly if such therapy can be initiated before or immediately after recognition of the first signs of this disease. The finding that the depletion of T cells from otherwise intact mice prevents the development of CM during P. berghei infection provides a model in which to analyze mechanisms by which these cells function in the pathogenesis of this disease.

View this table: [in this window] [in a new window]   TABLE 3.   CM in 0/0 mice infected with P. berghei ANKA