γδ T Cells but Not NK Cells Are Essential for Cell-Mediated Immunity against Plasmodium chabaudi Malaria

Blood-stage Plasmodium chabaudi infections are suppressed by antibody-mediated immunity and/or cell-mediated immunity (CMI). To determine the contributions of NK cells and γδ T cells to protective immunity, C57BL/6 (wild-type [WT]) mice and B-cell-deficient (J_H^−/−) mice were infected with P. chabaudi and depleted of NK cells or γδ T cells with monoclonal antibody. The time courses of parasitemia in NK-cell-depleted WT mice and J_H^−/− mice were similar to those of control mice, indicating that deficiencies in NK cells, NKT cells, or CD8⁺ T cells had little effect on parasitemia. In contrast, high levels of noncuring parasitemia occurred in J_H^−/− mice depleted of γδ T cells. Depletion of γδ T cells during chronic parasitemia in B-cell-deficient J_H^−/− mice resulted in an immediate and marked exacerbation of parasitemia, suggesting that γδ T cells have a direct killing effect in vivo on blood-stage parasites. Cytokine analyses revealed that levels of interleukin-10, gamma interferon (IFN-γ), and macrophage chemoattractant protein 1 (MCP-1) in the sera of γδ T-cell-depleted mice were significantly (P < 0.05) decreased compared to hamster immunoglobulin-injected controls, but these cytokine levels were similar in NK-cell-depleted mice and their controls. The time courses of parasitemia in CCR2^−/− and J_H^−/− × CCR2^−/− mice and in their controls were nearly identical, indicating that MCP-1 is not required for the control of parasitemia. Collectively, these data indicate that the suppression of acute P. chabaudi infection by CMI is γδ T cell dependent, is independent of NK cells, and may be attributed to the deficient IFN-γ response seen early in γδ T-cell-depleted mice.Malaria remains a leading cause of morbidity and mortality, annually killing about 2 million people worldwide (32, 33). Despite decades of research, malaria is a reemerging disease because of increasing drug resistance by malarial parasites and insecticide resistance by the mosquito vector. Most infected individuals do not succumb to malaria but develop clinical immunity where parasite replication is controlled to some degree by the immune system without eliciting clinical disease or sterile immunity (14, 38).Understanding the immunologic pathways leading to the control of blood-stage parasite replication is important for defining the mechanisms of disease pathogenesis and improving vaccines currently in development. The early events of the immune response depend upon activation of the innate immune system, which regulates the downstream adaptive immune response needed to control or cure (44). Natural killer (NK) and γδ T cells function early in the immune response to pathogens as components of the innate immune system. Both cell types have been proposed to play significant roles in the subsequent clearance of blood-stage malarial parasites by activating the adaptive immune system (35, 43, 44). The mechanism by which they accomplish this appears to be mediated via their secretion of gamma interferon (IFN-γ) induced by cytokines such as interleukin-12 (IL-12), tumor necrosis factor alpha (TNF-α), and IL-6 produced by other components of the innate immune system, including macrophages and dendritic cells (17, 25, 26, 37, 49).Blood-stage malaria parasites are cleared by mature isotypes of antibodies and/or by antibody-independent but T-cell-dependent mechanisms of immunity (2, 15, 22). Both responses require CD4⁺ αβ T cells; in addition, the expression of cell-mediated immunity (CMI) during both acute and chronic malaria is dependent on γδ T cells activated by CD4⁺ αβ T cells (29, 47, 49, 50). Wild-type (WT) mice depleted of γδ T cells by antibody treatment or gene knockout suppress P. chabaudi parasitemia by antibody-mediated immunity (AMI) (21, 52). Mice depleted of B cells by the same procedures also cure their acute infections in the same timeframe as intact control mice but then develop chronic low-grade parasitemia of long-lasting duration, indicating that B cells and their antibodies are needed to sterilize the infection as we originally reported (15, 48) and has since been confirmed by others (51). B-cell-deficient mice depleted of γδ T cells cannot suppress P. chabaudi parasitemia (49, 50, 52).The prominent role played by IFN-γ in immunity to malaria is generally accepted by most researchers. P. chabaudi malaria is more severe in WT mice treated with neutralizing antibody and in IFN-γ^−/− mice, as indicated by the increased magnitude and duration of parasitemia and mortality in mice deficient in IFN-γ versus intact controls (24, 39, 46). In B-cell-deficient animals, the similar neutralization of IFN-γ by treatment with anti-IFN-γ monoclonal antibody (MAb) or gene knockout of IFN-γ has an even greater effect on the time course of parasitemia, which remains at high levels and fails to cure (1, 46), indicating that IFN-γ is essential for the expression of anti-parasite CMI and contributes to AMI in this model system.The early source of IFN-γ remains controversial, with both NK cells and γδ T cells being proposed to produce this critical cytokine necessary for the activation of the adaptive immune response and the development of protective immunity (9). The results of earlier genetic studies failed to correlate susceptibility to P. chabaudi infection with NK activity (31, 44). Subsequently, Mohan et al. (25) reported that NK cell activity against tumor cell targets correlates with protection against P. chabaudi; anti-asialo GM1 polyclonal antibody depletion of NK cells results in significantly increased levels of peak parasitemia and a prolonged duration of infection compared to controls. The mode of action by which NK cells function appears to be via the secretion of cytokines (25) rather than direct cytotoxicity against the blood-stage parasites. The surface expression of lysosome-associated membrane protein 1 (LAMP-1) by subsets of human NK cells exposed to Plasmodium falciparum-infected erythrocytes may suggest otherwise (20). NK cells in collaboration with dendritic cells are responsible for optimal IFN-γ production dependent upon IL-12 (17, 36, 39, 40). In contrast to the findings of Mohan et al., other studies indicate similar P. chabaudi parasitemia in depleted mice and intact controls after NK1.1 MAb depletion of NK cells (19, 41, 53). Using microarray analysis of blood cells from P. chabaudi-infected mice, Kim et al. (18) reported a rapid production of IFN-γ and activation of IFN-γ-mediated signaling pathways as early as 8 h after infection; however, NK cells did not express IFN-γ or exhibit IFN-γ-mediated pathways in their analysis. At this time, NK cells are replicating and migrating from the spleen to the blood. In humans with P. falciparum malaria, increased production of IFN-γ by PBMC in response to parasitized RBCs correlates with protection from high-density parasitemia and clinical malaria (10, 11); early IFN-γ production by PBMC obtained from malaria naive donors is primarily by γδ T cells and not by NK cells (26). Animal models by definition do not exactly mimic the human condition, and the experimental malaria in mice uses distinct species from those that infect humans. Nevertheless, analysis of protective immunity provides important information on how a protective immune response to Plasmodium may be elicited.Whether both NK cells and γδ T cells have essential roles during the early stages of the immune response to blood-stage malaria remains to be determined. Likewise, whether these cells function early in CMI to malaria parasites is unknown. To address these issues, we infected NK-cell- or γδ-T-cell-depleted J_H^−/− mice with blood-stage P. chabaudi. The resulting time course of parasitemia was monitored and compared to control mice. In addition, spleen cells from depleted and control mice were profiled by cytofluorimetry, and the serum levels of inflammatory cytokines were measured.