The control of Plasmodium falciparum erythrocytic parasite density is essential for protection against malaria, because it prevents pathogenesis and progression toward severe disease. P falciparum blood-stage parasite cultures are inhibited by human V␥9V␦2 ␥␦ T cells, but the underlying mechanism remains poorly understood. Here, we show that both intraerythrocytic parasites and the extracellular red blood cell-invasive merozoites specifically activate V␥9V␦2 T cells in a ␥␦ T cell receptor-dependent manner and trigger their degranulation. In contrast, the ␥␦ T cell-mediated antiparasitic activity only targets the extracellular merozoites. Using perforin-deficient and granulysin-silenced T-cell lines, we demonstrate that granulysin is essential for the in vitro antiplasmodial process, whereas perforin is dispensable. Patients infected with P falciparum exhibited elevated granulysin plasma levels associated with high levels of granulysin-expressing V␦2 ؉ T cells endowed with parasite-specific degranulation capacity. This indicates in vivo activation of V␥9V␦2 T cells along with granulysin triggering and discharge during primary acute falciparum malaria. Altogether, this work identifies V␥9V␦2 T cells as unconventional immune effectors targeting the red blood cell-invasive extracellular P falciparum merozoites and opens novel perspectives for immune interventions harnessing the antiparasitic activity of V␥9V␦2 T cells to control parasite density in malaria patients. (Blood. 2011;118(26):6952-6962) IntroductionClinical malaria is associated with the intraerythrocytic asexual replication cycle of the Plasmodium sp parasite. Whereas young intraerythrocytic-stage parasites circulate in the blood, mature intraerythrocytic-stage parasites (trophozoites and schizonts) are sequestered in the microcirculation. On completion of intraerythrocytic development, extracellular invasive merozoites are released into the bloodstream, where they invade new red blood cells (RBCs), thus exponentially amplifying the density of blood-stage parasites. Control of parasite density is essential for protection against malaria, because it prevents pathogenesis and progression toward severe disease.Despite major research efforts, the immune mechanisms involved in the control of parasite biomass remain poorly understood. This lack of understanding impedes the rational development of immune-based interventions to prevent or cure malaria. Analysis of immune effectors that control blood-stage parasites has mainly focused on antibodydependent mechanisms, as passive transfer of immunoglobulins dramatically reduced parasite density in children with malaria. 1,2 Little attention has been paid to early immune responses that play, however, a pivotal role in the race between parasite development and the deployment of protective adaptive immune mechanisms. Recent studies have highlighted the role of innate immune effectors, including innate lymphocytes, in the early control of parasitemia before significant levels of specific antibodies are produced; howev...

␥␦ T cells have variously been implicated in the protection against, and the pathogenesis of, malaria, but few studies have examined the ␥␦ T-cell response to malaria in African children, who suffer the large majority of malaria-associated morbidity and mortality. This is unfortunate, since available data suggest that simple extrapolation of conclusions drawn from studies of nonimmune adults ex vivo and in vitro is not always possible. Here we show that both the frequencies and the absolute numbers of ␥␦ T cells are transiently increased following treatment of Plasmodium falciparum malaria in Ghanaian children and they can constitute 30 to 50% of all T cells shortly after initiation of antimalarial chemotherapy. The bulk of the ␥␦ T cells involved in this perturbation expressed V␦1 and had a highly activated phenotype. Analysis of the T-cell receptors (TCR) of the V␦1؉ cell population at the peak of their increase showed that all expressed V␥ chains were used, and CDR3 length polymorphism indicated that the expanded V␦1 population was highly polyclonal. A very high proportion of the V␦1 ؉ T cells produced gamma interferon, while fewer V␦1 ؉ cells than the average proportion of all CD3 ؉ cells produced tumor necrosis factor alpha. No interleukin 10 production was detected among TCR-␥␦ ؉ cells in general or V␦1 ؉ cells in particular. Taken together, our data point to an immunoregulatory role of the expanded V␦1 ؉ T-cell population in this group of semi-immune P. falciparum malaria patients.

TCR gamma delta(+) cells constitute <5% of all circulating T cells in healthy, adult Caucasians, and V(delta)1(+) cells constitute a minority of these cells. In contrast to TCR alpha beta(+) cells, their repertoire is selected extrathymically by environmental antigens. Although increased frequencies of V(delta)1(+) cells are found in several diseases, their function remains obscure. Here we show that the frequency of peripheral blood gamma delta T cells in healthy West Africans is about twice that of Caucasians, mainly due to a 5-fold increase in V(delta)1(+) cells, which is consequently the dominant subset. No age dependency of V(delta)1 frequencies was identified and the V(delta)1(+) cells in the African donors did not show preferential V(gamma) chain usage. Analysis of the CDR3 region size did not reveal any particular skewing of the V(delta)1 repertoire, although oligoclonality was more pronounced in adults compared to children. The proportions of CD8(+), CD38(+) and CD45RA(hi)CD45RO(-) cells within the V(delta)1(+) subset were higher in the African than in the European donors, without obvious differences in expression of activation markers. No significant correlations between levels of V(delta)1(+) cells and environmental antigens or immunological parameters were identified. Taken together, the evidence argues against a CDR3-restricted, antigen-driven expansion of V(delta)1(+) cells in the African study population. Our study shows that high frequencies of TCR gamma delta(+) cells with dominance of the V(delta)1(+) subset can occur at the population level in healthy people, raising questions about the physiological role of V(delta)1(+) T cells in the function and regulation of the immune system.

Malaria induces potent activation and expansion of the Vγ9Vδ2 subpopulation of γδT cells, which inhibit the Plasmodium falciparum blood cycle through soluble cytotoxic mediators, abrogating merozoite invasion capacity. Intraerythrocytic stages efficiently trigger Vγ9Vδ2 T-cell activation and degranulation through poorly understood mechanisms. P. falciparum blood-stage extracts are known to contain phosphoantigens able to stimulate Vγ9Vδ2 T cells, but how these are presented by intact infected red blood cells (iRBCs) remains elusive. Here we show that, unlike activation by phosphoantigen-expressing cells, Vγ9Vδ2 T-cell activation by intact iRBCs is independent of butyrophilin expression by the iRBC, and contact with an intact iRBC is not required. Moreover, blood-stage culture supernatants proved to be as potent activators of Vγ9Vδ2 T cells as iRBCs. Bioactivity in the microenvironment is attributable to phosphoantigens, as it is dependent on the parasite DOXP pathway, on Vγ9Vδ2 TCR signaling, and on butyrophilin expression by Vγ9Vδ2 T cells. Kinetic studies showed that the phosphoantigens were released at the end of the intraerythrocytic cycle at the time of parasite egress. We document exquisite sensitivity of Vγ9Vδ2 T cells, which respond to a few thousand parasites. These data unravel a novel framework, whereby release of phosphoantigens into the extracellular milieu by sequestered parasites likely promotes activation of distant Vγ9Vδ2 T cells that in turn exert remote antiparasitic functions.