Unique T cell effector functions elicited by Plasmodium falciparum epitopes in malaria-exposed Africans tested by three T cell assays

Natural immunity to malaria is characterized by low level CD4 T cell reactivity detected by either lymphoproliferation or IFN-gamma secretion. Here we show a doubling in the detection rate of responders to the carboxyl terminus of circumsporozoite protein (CS) of Plasmodium falciparum by employing three T cell assays simultaneously: rapid IFN-gamma secretion (ex vivo ELISPOT), IFN-gamma secretion after reactivation of memory T cells and expansion in vitro (cultured ELISPOT), and lymphoproliferation. Remarkably, for no individual peptide did a positive response for one T cell effector function correlate with any other. Thus these CS epitopes elicited unique T cell response patterns in malaria-exposed donors. Novel or important epitope responses may therefore be missed if only one T cell assay is employed. A borderline correlation was found between anti-CS Ab levels and proliferative responses, but no correlation was found with ex vivo or cultured IFN-gamma responses. This suggested that the proliferating population, but not the IFN-gamma-secreting cells, contained cells that provide help for Ab production. The data suggest that natural immunity to malaria is a complex function of T cell subgroups with different effector functions and has important implications for future studies of natural T cell immunity.     

Persistent cytomegalovirus-specific memory responses in the lung allograft and blood following primary infection in lung transplant recipients

Primary CMV infection in lung transplant recipients (LTRs) is associated with increased mortality. We studied 22 donor CMV-positive, recipient-negative (D(+)R(-)) LTRs for the development of posttransplant CMV-specific immunity. We found that 13 of 22 D(+)R(-) LTRs (59.1%) seroconverted (CMV IgG Ab(+)). Using pooled peptides of the immunodominant CMV Ags pp65 and IE1, we detected CMV-specific CD8(+)IFN-gamma(+) T cells in the PBMC of 90% of seroconverted individuals following primary infection by intracellular cytokine staining. In contrast, few seroconverters had detectable CMV-specific CD4(+)IFN-gamma(+) T cells during viral latency. However, the majority of IgG(+) LTRs demonstrated CMV-specific CD4(+) and CD8(+) T cell proliferative responses from PBMC, with CD4(+)IFN-gamma(+) T cells detectable upon re-expansion. Examination of lung allograft mononuclear cells obtained by bronchoalveolar lavage revealed both CMV-specific CD4(+) and CD8(+)IFN-gamma(+) T cells, including patients from whom CD4(+)IFN-gamma(+) T cells were simultaneously undetectable in the PBMC, suggesting differential effector memory populations between these compartments. Moreover, both responses in the PBMC and lung allograft were found to persist, despite substantial immunosuppression, long after primary infection. Clinical correlation in this cohort demonstrated that the acquisition of CMV immunity was associated with freedom from CMV disease (p < or = 0.009) and preservation of allograft function (p < or = 0.02) compared with those who failed to develop CMV immunity. Together, our data reveal immunologic heterogeneity in D(+)R(-) LTRs, with the development and persistence of primary CMV responses that may provide clinical benefit.     

Interferon-gamma responses to Plasmodium falciparum liver-stage antigen-1 and merozoite-surface protein-1 increase with age in children in a malaria holoendemic area of western Kenya

BACKGROUND: In areas of high-level, year-round malaria transmission, morbidity and mortality due to malaria decrease after the first two to three years of life. This reduction may be related to the development of cellular immunity to specific antigens expressed in the different life-cycle stages of Plasmodium falciparum. METHODS: A cross sectional study was conducted to evaluate T cell cytokine responses to the P. falciparum pre-erythrocytic antigen liver-stage antigen-1 (LSA-1) and the blood-stage antigen merozoite-surface protein-1 (MSP-1) in children under five years of age residing in a malaria holoendemic region of western Kenya. Interferon-gamma (IFN-gamma) and interleukin-10 (IL-10) responses to the LSA-1 T3 peptide (aa 1813-1835) and the MSP-1 aa20-39 peptide were tested in 48 children. RESULTS: The proportion of children producing IFN-gamma to LSA-1 and to MSP-1 increased with age: in the 0-12, 13-24, 25-36 and 37-48 month age groups, zero, 11.1, 36.4 and 40% of children had IFN-gamma responses to LSA-1 (p = 0.019), and 10, 10, 27.7 and 40% of children had IFN-gamma responses to MSP-1 (p = 0.07), respectively. In contrast, the proportion of children producing IL-10 to LSA-1 and MSP-1 was similar in all age groups. CONCLUSION: The data suggest that development of IFN-gamma responses to LSA-1 and MSP-1 requires increased age and/or repeated exposure, whereas IL-10 responses to these antigens may occur at any age and with limited exposure. The data also demonstrate that by the age of 4 years, children in a malaria holoendemic area develop frequencies of IFN-gamma responses to LSA-1 and MSP-1 similar to those seen in adults in the area.     

The complexity of protective immunity against liver-stage malaria

Sterile protective immunity against challenge with Plasmodium spp. sporozoites can be induced in multiple model systems and humans by immunization with radiation-attenuated Plasmodium spp. sporozoites. The infected hepatocyte has been established as the primary target of this protection, but the underlying mechanisms have not been completely defined. Abs, CD8+ T cells, CD4+ T cells, cytokines (including IFN-gamma and IL-12), and NO have all been implicated as critical effectors. Here, we have investigated the mechanisms of protective immunity induced by immunization with different vaccine delivery systems (irradiated sporozoites, plasmid DNA, synthetic peptide/adjuvant, and multiple Ag peptide) in genetically distinct inbred strains, genetically modified mice, and outbred mice. We establish that there is a marked diversity of T cell-dependent immune responses that mediate sterile protective immunity against liver-stage malaria. Furthermore, we demonstrate that distinct mechanisms of protection are induced in different strains of inbred mice by a single method of immunization, and in the same strain by different methods of immunization. These data underscore the complexity of the murine host response to a parasitic infection and suggest that an outbred human population may behave similarly. Data nevertheless suggest that a pre-erythrocytic-stage vaccine should be designed to induce CD8+ T cell- and IFN-gamma-mediated immune responses and that IFN-gamma responses may represent an in vitro correlate of pre-erythrocytic-stage protective immunity.     

Protective immunity induced with malaria vaccine, RTS,S, is linked to Plasmodium falciparum circumsporozoite protein-specific CD4+ and CD8+ T cells producing IFN-gamma

The Plasmodium falciparum circumsporozoite (CS) protein-based pre-erythrocytic stage vaccine, RTS,S, induces a high level of protection against experimental sporozoite challenge. The immune mechanisms that constitute protection are only partially understood, but are presumed to rely on Abs and T cell responses. In the present study we compared CS protein peptide-recalled IFN-gamma reactivity of pre- and RTS,S-immune lymphocytes from 20 subjects vaccinated with RTS,S. We observed elevated IFN-gamma in subjects protected by RTS,S; moreover, both CD4(+) and CD8(+) T cells produced IFN-gamma in response to CS protein peptides. Significantly, protracted protection, albeit observed only in two of seven subjects, was associated with sustained IFN-gamma response. This is the first study demonstrating correlation in a controlled Plasmodia sporozoite challenge study between protection induced by a recombinant malaria vaccine and Ag-specific T cell responses. Field-based malaria vaccine studies are in progress to validate the establishment of this cellular response as a possible in vitro correlate of protective immunity to exo-erythrocytic stage malaria vaccines.     

Selectively impaired CD8+ but not CD4+ T cell cycle arrest during priming as a consequence of dendritic cell interaction with plasmodium-infected red cells

Individuals living in malaria-endemic areas show generally low T cell responses to malaria Ags. In this study, we show murine dendritic cell (DC) interaction with parasitized erythrocytes (pRBC) arrested their maturation, resulting in impaired ability to stimulate naive, but not recall T cell responses in vitro and in vivo. Moreover, within the naive T cell population, pRBC-treated DC were selectively deficient in priming CD8(+) but not CD4(+) T cells. Indeed, DC that had taken up pRBC were shown for the first time to efficiently prime CD4(+) T cell responses to a known protective merozoite Ag, MSP4/5. In contrast, impaired priming resulted in decreases in both proliferation and cytokine production by CD8(+) T cells. Deficient priming was observed to both a model and a Plasmodium berghei-specific CD8(+) T cell epitope. The mechanisms underlying the inability of parasite-treated DC to prime CD8(+) T cells were explored. pRBC treatment of DC from wild-type C57BL/6, but not from IL-10 knockout animals, suppressed DC-mediated T cell priming across a Transwell, suggesting active IL-10-dependent suppression. CD8(+) T cells were arrested at the G(0) stage of the cell cycle after two cell divisions post-Ag stimulation. The proliferation arrest was partially reversible by the addition of IL-2 or IL-7 to responder cultures. These results suggest that in malaria-endemic areas, priming of CD8(+) T cell responses may be more difficult to induce via vaccination than the priming of CD4(+) T cells. Moreover, pathogens may selectively target the CD8(+) T cell arm of protective immunity for immune evasion.     

Longevity and composition of cellular immune responses following experimental Plasmodium falciparum malaria infection in humans

Cellular responses to Plasmodium falciparum parasites, in particular interferon-gamma (IFNγ) production, play an important role in anti-malarial immunity. However, clinical immunity to malaria develops slowly amongst naturally exposed populations, the dynamics of cellular responses in relation to exposure are difficult to study and data about the persistence of such responses are controversial. Here we assess the longevity and composition of cellular immune responses following experimental malaria infection in human volunteers. We conducted a longitudinal study of cellular immunological responses to sporozoites (PfSpz) and asexual blood-stage (PfRBC) malaria parasites in na?ve human volunteers undergoing single (n?=?5) or multiple (n?=?10) experimental P. falciparum infections under highly controlled conditions. IFNγ and interleukin-2 (IL-2) responses following in vitro re-stimulation were measured by flow-cytometry prior to, during and more than one year post infection. We show that cellular responses to both PfSpz and PfRBC are induced and remain almost undiminished up to 14 months after even a single malaria episode. Remarkably, not only 'adaptive' but also 'innate' lymphocyte subsets contribute to the increased IFNγ response, including αβT cells, γδT cells and NK cells. Furthermore, results from depletion and autologous recombination experiments of lymphocyte subsets suggest that immunological memory for PfRBC is carried within both the αβT cells and γδT compartments. Indeed, the majority of cytokine producing T lymphocytes express an CD45RO(+) CD62L(-) effector memory (EM) phenotype both early and late post infection. Finally, we demonstrate that malaria infection induces and maintains polyfunctional (IFNγ(+)IL-2(+)) EM responses against both PfRBC and PfSpz, previously found to be associated with protection. These data demonstrate that cellular responses can be readily induced and are long-lived following infection with P. falciparum, with a persisting contribution by not only adaptive but also (semi-)innate lymphocyte subsets. The implications hereof are positive for malaria vaccine development, but focus attention on those factors potentially inhibiting such responses in the field.     

Long-Lived Antibody and B Cell Memory Responses to the Human Malaria Parasites,Plasmodium falciparum and Plasmodium vivax

Antibodies constitute a critical component of the naturally acquired immunity that develops following frequent exposure to malaria. However, specific antibody titres have been reported to decline rapidly in the absence of reinfection, supporting the widely perceived notion that malaria infections fail to induce durable immunological memory responses. Currently, direct evidence for the presence or absence of immune memory to malaria is limited. In this study, we analysed the longevity of both antibody and B cell memory responses to malaria antigens among individuals who were living in an area of extremely low malaria transmission in northern Thailand, and who were known either to be malaria naïve or to have had a documented clinical attack of P. falciparum and/or P. vivax in the past 6 years. We found that exposure to malaria results in the generation of relatively avid antigen-specific antibodies and the establishment of populations of antigen-specific memory B cells in a significant proportion of malaria-exposed individuals. Both antibody and memory B cell responses to malaria antigens were stably maintained over time in the absence of reinfection. In a number of cases where antigen-specific antibodies were not detected in plasma, stable frequencies of antigen-specific memory B cells were nonetheless observed, suggesting that circulating memory B cells may be maintained independently of long-lived plasma cells. We conclude that infrequent malaria infections are capable of inducing long-lived antibody and memory B cell responses.     

Immune regulation of protection and pathogenesis in Plasmodium falciparum malaria

The immune mechanisms whereby malaria parasites are eliminated by the human host or how they may avoid the immune response are poorly understood. Individuals living in malaria-endemic areas gradually acquire immunity. It is well established that this immunity involves both cell-mediated and humoral mechanisms and that T cells are the major regulators in both these events. The existence of functionally distinct P. falciparum-specific CD4+ T-cell subsets in humans has been shown in several studies. However, in contrast to what is the case in murine models there is no definitive link between the activation of various T cells and the course of human P. falciparum blood-stage infection. In the present paper we will review recent findings which illustrate how the balance between functionally different T-cell subsets affects the development of malaria immunity but also may contribute to its pathogenicity. An example of the latter is the deposition of IgE-containing immune complexes in small vessels, probably leading to local overproduction of tumor-necrosis factor (TNF), a pathogenic factor in malaria.     

Naturally acquired immune responses against Plasmodium falciparum sporozoites and liver infection

Malaria is a vector-borne infectious disease caused by infection with eukaryotic pathogens termed Plasmodium. Epidemiological hallmarks of Plasmodium falciparum malaria are continuous re-infections, over which time the human host may experience several clinical malaria episodes, slow acquisition of partial protection against infection, and its partial decay upon migration away from endemic regions. To overcome the exposure-dependence of naturally acquired immunity and rapidly elicit robust long-term protection are ultimate goals of malaria vaccine development. However, cellular and molecular correlates of naturally acquired immunity against either parasite infection or malarial disease remain elusive. Sero-epidemiological studies consistently suggest that acquired immunity is primarily directed against the asexual blood stages. Here, we review available data on the relationship between immune responses against the Anopheles mosquito-transmitted sporozoite and exo-erythrocytic liver stages and the incidence of malaria. We discuss current limitations and research opportunities, including the identification of additional sporozoite antigens and the use of systematic immune profiling and functional studies in longitudinal cohorts to look for pre-erythrocytic signatures of naturally acquired immunity.     

A retrospective evaluation of the role of T cells in the development of malaria vaccine

Due to the fact that the life cycle of malaria parasites is complex, undergoing both an extracellular and intracellular phases in its host, the human immune system has to mobilize both the humoral and cellular arms of immune responses to fight against this parasitic infection. Whereas humoral immunity is directed toward the extracellular stages which include sporozoites and merozoites, cell-mediated immunity (CMI), in which T cells play a major role, targets hepatic stages - liver stages - of the parasites. In this review, the role of T cells in protective immunity against liver stages of the malaria infection is being re-evaluated. Furthermore, this review intends to address how to translate the findings regarding the role of T cells obtained in experimental systems to actual development of malaria vaccine for humans.     

Plasmodium falciparum liver-stage antigen-1 peptide-specific interferon-gamma responses are not suppressed during uncomplicated malaria in African children.

Liver-stage antigen (LSA)-1 is a candidate vaccine molecule for Plasmodium falciparum malaria, but knowledge of the evolution of naturally acquired immune responses to LSA-1 in African children is lacking. We therefore assessed cellular immune responses to two defined T cell epitopes of LSA-1, during and after uncomplicated P. falciparum malaria in a group of Gabonese children. In terms of their prevalence, interferon (IFN)-gamma responses of peripheral blood mononuclear cells (PBMC) to an LSA-1 N-terminal peptide, T1, were significantly higher when measured during the acute phase compared with convalescence. IFN-gamma responses to the LSA-J (hinge region) peptide showed a similar profile, but at a lower prevalence. Depletion experiments confirmed that CD8+ T cells are a major source of peptide-driven IFN-gamma, but both lymphoproliferation and the production of IL-10 in response to either of the peptides was low in all children at all times. PBMC from 25% of the children failed to produce IFN-gamma in response to either peptide at any time-point. The results suggest that lymphocytes producing IFN-gamma in response to at least one T cell epitope of LSA-1 are most frequent in the peripheral circulation during the acute phase of P. falciparum malaria. Thus, in this case, the generalised suppression of cell-mediated responses which characterises acute malaria does not affect liver-stage antigen-specific IFN-gamma production. These findings imply that measurements of the frequency of parasite antigen-specific cellular immune responses in clinically healthy individuals may represent significant underestimations, which has important implications for the design of field-based vaccine antigen-related studies.     

Acquired immune responses to Plasmodium falciparum merozoite surface protein-1 in the human fetus.

Infants born in areas of stable malaria transmission are relatively protected against severe morbidity and high density Plasmodium falciparum blood-stage infection. This protection may involve prenatal sensitization and immunologic reactivity to malaria surface ligands that participate in invasion of red cells. We examined cord blood T and B cell immunity to P. falciparum merozoite surface protein-1 (MSP-1) in infants born in an area of stable malaria transmission in Kenya. T cell cytokine responses to the C-terminal 19-kDa fragment of MSP-1 (MSP-1(19)) were detected in 24 of 92 (26%) newborns (4-192 IFN-gamma and 3-88 IL-4-secreting cells per 10(6)/cord blood lymphocytes). Peptide epitopes in the N-terminal block 3 region of MSP-1 also drove IFN-gamma and/or IL-13 production. There was no evidence of prenatal T cell sensitization to liver-stage Ag-1. A total of 5 of 86 (6%) newborns had cord blood anti-MSP-1(19) IgM Abs, an Ig isotype that does not cross the placenta and is therefore of fetal origin. The frequency of neonatal B cell sensitization was higher than that indicated by serology alone, as 5 of 27 (18%) cord blood samples contained B cells that produced IgG when stimulated with MSP-1(19) in vitro. Neonatal B cell IgG responses were restricted to the Q-KNG allele of MSP-1(19), the major variant in this endemic area, whereas T cells responded to all four MSP-1(19) alleles evaluated. In utero sensitization to MSP-1 correlated with the presence of malaria parasites in cord blood (chi(2) = 20, p < 0.0001). These data indicate that prenatal sensitization to blood-stage Ags occurs in infants born in malaria endemic areas.     

IL-12 and NK cells are required for antigen-specific adaptive immunity against malaria initiated by CD8+ T cells in the Plasmodium yoelii model.

CD8+ T cells have been implicated as critical effector cells in protection against preerythrocytic stage malaria, including the potent protective immunity of mice and humans induced by immunization with radiation-attenuated Plasmodium spp. sporozoites. This immunity is directed against the Plasmodium spp. parasite developing within the host hepatocyte and for a number of years has been presumed to be mediated directly by CD8+ CTL or indirectly by IFN-gamma released from CD8+ T cells. In this paper, in BALB/c mice, we establish that after immunization with irradiated sporozoites or DNA vaccines parasite-specific CD8+ T cells trigger a novel mechanism of adaptive immunity that is dependent on T cell- and non-T cell-derived cytokines, in particular IFN-gamma and IL-12, and requires NK cells but not CD4+ T cells. The absolute requirement for CD8+ T cells to initiate such an effector mechanism, and the requirement for IL-12 and NK cells in such vaccine-induced protective immunity, are unique and underscore the complexity of the immune responses that protect against malaria and other intracellular pathogens.     

Anti-CD25 antibody enhancement of vaccine-induced immunogenicity: increased durable cellular immunity with reduced immunodominance

An efficacious vaccine strategy must be capable of inducing strong responses of an appropriate phenotype that are long lasting and sufficiently broad to prevent pathogen escape mechanisms. In the present study, we use anti-CD25 mAb to augment vaccine-induced immunity in mice. We demonstrate that coformulation of Ab and poxviral- or adenoviral-vectored vaccines induces significantly increased T cell responses to a malaria Ag; prior anti-CD25 Ab administration was not required for this effect. Furthermore, this vaccination approach subverts immunodominant epitope hierarchies by enhancing responses to subdominant epitopes induced by recombinant modified vaccinia virus Ankara immunization. Administration of anti-CD25 with a vaccine also induces more durable immunity compared with vaccine alone; significantly higher T cell responses were observed 100 days after the primary immunization. Enhanced immunogenicity is observed for multiple vaccine types with enhanced CD4+ and CD8+ T cell responses induced by bacillus Calmette-Guérin and a recombinant subunit protein vaccine to hepatitis B virus and with multiple Ags of tumor, viral, bacterial, and parasitic origin. Vaccine strategies incorporating anti-CD25 lead to improved protection against pre-erythrocytic malaria challenge. These data underpin new strategies for the design and development of more efficacious vaccines in clinical settings.     

Transmission intensity determines lymphocyte responsiveness and cytokine bias in human lymphatic filariasis

Humans living in areas where filariasis is endemic vary greatly in their exposure to mosquito-borne infective third-stage larvae (L3) of these parasitic helminths. Because the intensity of exposure to Ags affects T cell differentiation and susceptibility to parasitic infections in murine models, we compared T cell and cytokine responses in 97 residents of two villages in Papua New Guinea, where transmission intensity of Wuchereria bancrofti differed by 63-fold (37 vs 2355 L3 per person per year). Residents of the high transmission village had 4- to 11-fold lower proliferation and IFN-gamma responses to filarial Ags, nonparasite Ag, and PHA by PBMC compared with the low transmission village (p < 0.01) even when subjects were matched for intensity of infection. In contrast, filarial Ag-driven IL-5 production was 5.5-fold greater (p < 0.001), and plasma IL-4 and TGF-beta levels were 4-fold and 34% higher, respectively, in residents of the high transmission village. IL-4 and IL-10 responses by PBMC differed little according to village, and increased production of the counterregulatory cytokines IL-10 or TGF-beta by PBMC did not correlate with weak proliferation and IFN-gamma responses. Plasma IL-5, IFN-gamma, and IL-10 levels were similar in the two villages. These data demonstrate that the intensity of exposure to L3 affects lymphocyte responsiveness and cytokine bias possibly by a mechanism that alters APC function.     

Immune response to pre-erythrocytic stages of malaria parasites

Immunization with radiation-attenuated Plasmodium spp. sporozoites induces sterile protective immunity against parasite challenge. This immunity is targeted primarily against the intrahepatic parasite and appears to be sustained long term even in the absence of sporozoite exposure. It is mediated by multifactorial mechanisms, including T cells directed against parasite antigens expressed in the liver stage of the parasite life cycle and antibodies directed against sporozoite surface proteins. In rodent models, CD8+ T cells have been implicated as the principal effector cells, and IFN-gamma as a critical effector molecule. IL-4 secreting CD4+ T cells are required for induction of the CD8+ T cell responses, and Th1 CD4+ T cells provide help for optimal CD8+ T cell effector activity. Components of the innate immune system, including gamma-delta T cells, natural killer cells and natural killer T cells, also play a role. The precise nature of pre-erythrocytic stage immunity in humans, including the contribution of these immune responses to the age-dependent immunity naturally acquired by residents of malaria endemic areas, is still poorly defined. The importance of immune effector targets at the pre-erythrocytic stage of the parasite life cycle is highlighted by the fact that infection-blocking immunity in humans rarely, if ever, occurs under natural conditions. Herein, we review our current understanding of the molecular and cellular aspects of pre-erythrocytic stage immunity.     

The dendritic cell-specific chemokine,dendritic cell-derived CC chemokine 1, enhances protective cell-mediated immunity to murine malaria

Cell-mediated immunity plays a crucial role in the control of many infectious diseases, necessitating the need for adjuvants that can augment cellular immune responses elicited by vaccines. It is well established that protection against one such disease, malaria, requires strong CD8(+) T cell responses targeted against the liver stages of the causative agent, Plasmodium spp. In this report we show that the dendritic cell-specific chemokine, dendritic cell-derived CC chemokine 1 (DC-CK1), which is produced in humans and acts on naive lymphocytes, can enhance Ag-specific CD8(+) T cell responses when coadministered with either irradiated Plasmodium yoelii sporozoites or a recombinant adenovirus expressing the P. yoelii circumsporozoite protein in mice. We further show that these enhanced T cell responses result in increased protection to malaria in immunized mice challenged with live P. yoelii sporozoites, revealing an adjuvant activity for DC-CK1. DC-CK1 appears to act preferentially on naive mouse lymphocytes, and its adjuvant effect requires IL-12, but not IFN-gamma or CD40. Overall, our results show for the first time an in vivo role for DC-CK1 in the establishment of primary T cell responses and indicate the potential of this chemokine as an adjuvant for vaccines against malaria as well as other diseases in which cellular immune responses are important.     

Innate immune response to malaria: rapid induction of IFN-gamma from human NK cells by live Plasmodium falciparum-infected erythrocytes

To determine the potential contribution of innate immune responses to the early proinflammatory cytokine response to Plasmodium falciparum malaria, we have examined the kinetics and cellular sources of IFN-gamma production in response to human PBMC activation by intact, infected RBC (iRBC) or freeze-thaw lysates of P. falciparum schizonts. Infected erythrocytes induce a more rapid and intense IFN-gamma response from malaria-naive PBMC than do P. falciparum schizont lysates correlating with rapid iRBC activation of the CD3(-)CD56(+) NK cell population to produce IFN-gamma. IFN-gamma(+) NK cells are detectable within 6 h of coculture with iRBC, their numbers peaking at 24 h in most donors. There is marked heterogeneity between donors in magnitude of the NK-IFN-gamma response that does not correlate with mitogen- or cytokine-induced NK activation or prior malaria exposure. The NK cell-mediated IFN-gamma response is highly IL-12 dependent and appears to be partially IL-18 dependent. Exogenous rIL-12 or rIL-18 did not augment NK cell IFN-gamma responses, indicating that production of IL-12 and IL-18 is not the limiting factor explaining differences in NK cell reactivity between donors or between live and dead parasites. These data indicate that NK cells may represent an important early source of IFN-gamma, a cytokine that has been implicated in induction of various antiparasitic effector mechanisms. The heterogeneity of this early IFN-gamma response between donors suggests a variation in their ability to mount a rapid proinflammatory cytokine response to malaria infection that may, in turn, influence their innate susceptibility to malaria infection, malaria-related morbidity, or death from malaria.