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.

BackgroundSevere malarial anaemia (SMA) is a major life-threatening complication of paediatric malaria. Protracted production of pro-inflammatory cytokines promoting erythrophagocytosis and depressing erythropoiesis is thought to play an important role in SMA, which is characterized by a high TNF/IL-10 ratio. Whether this TNF/IL-10 imbalance results from an intrinsic incapacity of SMA patients to produce IL-10 or from an IL-10 unresponsiveness to infection is unknown. Monocytes and T cells are recognized as the main sources of TNF and IL-10 in vivo, but little is known about the activation status of those cells in SMA patients.MethodsThe IL-10 and TNF production capacity and the activation phenotype of monocytes and T cells were compared in samples collected from 332 Ghanaian children with non-overlapping SMA (n = 108), cerebral malaria (CM) (n = 144) or uncomplicated malaria (UM) (n = 80) syndromes. Activation status of monocytes and T cells was ascertained by measuring HLA-DR+ and/or CD69+ surface expression by flow cytometry. The TNF and IL-10 production was assessed in a whole-blood assay after or not stimulation with lipopolysaccharide (LPS) or phytohaemaglutinin (PHA) used as surrogate of unspecific monocyte and T cell stimulant. The number of circulating pigmented monocytes was also determined.ResultsMonocytes and T cells from SMA and CM patients showed similar activation profiles with a comparable decreased HLA-DR expression on monocytes and increased frequency of CD69+ and HLA-DR+ T cells. In contrast, the acute-phase IL-10 production was markedly decreased in SMA compared to CM (P = .003) and UM (P = .004). Although in SMA the IL-10 response to LPS-stimulation was larger in amplitude than in CM (P = .0082), the absolute levels of IL-10 reached were lower (P = .013). Both the amplitude and levels of TNF produced in response to LPS-stimulation were larger in SMA than CM (P = .019). In response to PHA-stimulation, absolute levels of IL-10 produced in SMA were lower than in CM (P = .005) contrasting with TNF levels, which were higher (P = .001).ConclusionsThese data reveal that SMA patients have the potential to mount efficient IL-10 responses and that the TNF/IL-10 imbalance may reflect a specific monocyte and T cell programming/polarization pattern in response to infection.

During Plasmodium falciparum infections, erythrocyte-stage parasites inhibit dendritic cell maturation and function, compromising effective antimalarial adaptive immunity. Human Vγ9Vδ2 T cells can act in vitro as antigen-presenting cells (APCs) and induce αβ T-cell activation. However, the relevance of this activity in vivo has remained elusive. Because Vγ9Vδ2 T cells are activated during the early immune response against P. falciparum infection, we investigated whether they could contribute to the instruction of adaptive immune responses toward malaria parasites. In P. falciparum-infected patients, Vγ9Vδ2 T cells presented increased surface expression of APC-associated markers HLA-DR and CD86. In response to infected red blood cells in vitro, Vγ9Vδ2 T cells upregulated surface expression of HLA-DR, HLA-ABC, CD40, CD80, CD83, and CD86, induced naive αβ T-cell responses, and cross- presented soluble prototypical protein to antigen-specific CD8+ T cells. Our findings qualify Vγ9Vδ2 T cells as alternative APCs, which could be harnessed for therapeutic interventions and vaccine design.

Abstract;D T cells recognize stress-induced autoantigens and contribute to immunity against infections and cancer. Our previous study revealed that VD2-negative ( neg ) ;D T lymphocytes isolated from transplant recipients infected by cytomegalovirus (CMV) killed both CMV-infected cells and HT29 colon cancer cells in vitro. To investigate the antitumor effects of VD2 neg clones in vivo, we generated hypodermal HT29 tumors in immunodeficient mice. Concomitant injections of VD2 neg clones, in contrast to VD2 + cells, prevented the development of HT29 tumors. VD2 neg clones expressed chemokine C-C motif receptor 3 (CCR3) and migrated in vitro in response to chemokines secreted by HT29 cells, among which were the CCR3 ligands macrophage inflammatory protein-1D and monocyte chemoattractant protein-4. More importantly, a systemic i.p. treatment with VD2 neg clones delayed the growth of HT29 s.c. tumors. The effect of in vivo ;D T-cell passive immunotherapy on tumor growth could be reverted by addition of a blocking anti-CCR3 antibody. ;D T-cell passive immunotherapy was dependent on the cytotoxic activity of the ;D effectors toward their targets because VD2 neg clones were not able to inhibit the growth of A431 hypodermal tumors. Our findings suggest that CMV-specific VD2 neg cells could target in vivo cancer cells, making them an attractive candidate for antitumor immunotherapy. [Cancer Res 2009;69(9):3971-8]

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