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The effect of declining exposure on T cell-mediated immunity to Plasmodium falciparum – an epidemiological “natural experiment”
© The Author(s). 2016
Received: 27 May 2016
Accepted: 31 August 2016
Published: 22 September 2016
Naturally acquired immunity to malaria may be lost with lack of exposure. Recent heterogeneous reductions in transmission in parts of Africa mean that large populations of previously protected people may lose their immunity while remaining at risk of infection.
Using two ethnically similar long-term cohorts of children with historically similar levels of exposure to Plasmodium falciparum who now experience very different levels of exposure, we assessed the effect of decreased parasite exposure on antimalarial immunity. Peripheral blood mononuclear cells (PBMCs) from children in each cohort were stimulated with P. falciparum and their P. falciparum-specific proliferative and cytokine responses were compared.
We demonstrate that, while P. falciparum-specific CD4+ T cells are maintained in the absence of exposure, the proliferative capacity of these cells is altered considerably. P. falciparum-specific CD4+ T cells isolated from children previously exposed, but now living in an area of minimal exposure (“historically exposed”) proliferate significantly more upon stimulation than cells isolated from children continually exposed to the parasite. Similarly, PBMCs from historically exposed children expressed higher levels of pro-inflammatory cytokines and lower levels of anti-inflammatory cytokines after stimulation with P. falciparum. Notably, we found a significant positive association between duration since last febrile episode and P. falciparum-specific CD4+ T cell proliferation, with more recent febrile episodes associated with lower proliferation.
Considered in the context of existing knowledge, these data suggest a model explaining how immunity is lost in absence of continuing exposure to P. falciparum.
Malaria remains a significant threat to human health. While it is difficult to estimate attributable numbers of cases and deaths, the recent WHO report suggests at least 198 million cases were reported in 2013, leading to approximately 584,000 deaths . A large proportion of this burden is borne by Africa, where despite massive investments in malaria control and prevention, 57 % of the population continue to live in areas of moderate to intense malaria transmission .
Studies in malaria endemic areas consistently demonstrate that incidence and severity of disease decreases significantly with age, indicating that individuals living in these areas acquire a degree of immunity to clinical malaria. This protection is, however, only acquired following repeated infections and is not sterile [3, 4]. Although the mechanisms that underpin naturally acquired immunity to malaria (NAI) remain poorly understood, a review of literature suggests that it is comprised of two main components, namely (1) an anti-parasite component resulting in control of parasite replication and parasite clearance [5, 6] and (2) the ability to tolerate parasites asymptomatically. The latter component is likely to include an immunoregulatory (immune tolerance) element that contributes to control of symptoms and clinical immunity [7, 8]. Antibody-dependent mechanisms play a major role in parasite control and clearance , with contributions from innate and cellular arms of immunity . In contrast, the mechanism for the establishment of Plasmodium falciparum-specific immune “tolerance” is less understood. Recent studies suggest that repeated exposure to P. falciparum, as experienced in areas of high malaria endemicity, is required for the establishment of tolerance , which may be associated with the loss and/or altered function of several immune cell types, including γδ T cells , αβ T cells [9–12], B cells , and myeloid cells . Repeated exposure also results in an expansion of “self-regulating” Th1 cells [7, 15, 16], which produce IL-10 in combination with IFN-γ. IL-10 is a key immunomodulatory cytokine and plays an important role in mouse models of malaria [17–19]. Continual exposure to P. falciparum therefore appears to result in modulation of inflammation and associated immunopathology through regulation at multiple levels of the immune system.
Unfortunately, individuals who acquire immunity in malaria endemic areas but then migrate to malaria-free regions for prolonged periods appear to be susceptible to clinical disease upon returning to the endemic region [20–22]. This loss of immune protection may reflect defective antimalarial immunity related to poor induction and maintenance of long-lived memory responses . However, while protective plasma antibody levels decay rapidly (especially in young children) [23, 24], rapid boosting of antibody responses to a number of P. falciparum antigens have been reported upon re-exposure following periods of no exposure [25, 26]. In agreement with this, we have previously demonstrated that, while P. falciparum-specific antibody levels fall to undetectable levels in the absence of persistent P. falciparum exposure, P. falciparum-specific memory B cells are maintained at similar levels to those found in children continually exposed to the parasite . Furthermore, one might also infer a loss of tolerance to parasitemia from observations of the cytokine profiles of previously immune, returning travelers  and from the fact that these individuals appear to become unwell at parasite densities that they might previously have tolerated asymptomatically .
The mechanism(s) by which P. falciparum-specific T cell memory is induced and maintained is poorly understood. While some reports have described impairments in the establishment of P. falciparum-specific T cell memory (stemming from antigenic diversity , infection-related depletion of antigen-specific T cells [31, 32], and impaired dendritic cells ), animal models suggest that P. falciparum-specific memory T cell populations are maintained normally after infection. To date, only one study has directly assessed the longevity of P. falciparum-specific T cell responses in humans following a period of minimal exposure. This study measured P. falciparum-specific responses in adults living in an area of low malaria endemicity in Northern Thailand and demonstrated that, while some antimalarial T cell responses (IFN-γ producing T cells) were relatively short-lived, others (IL-10 producing T cells) were maintained for much longer in the absence of exposure . The limitation of this study, however, was that the majority of the malaria-exposed individuals had experienced only one documented episode of malaria in their lifetime. Given the previously described role that endemic malaria has in shaping the P. falciparum-specific T cell response [8–12], it is reasonable to suspect that the longevity of memory responses established in individuals previously living in malaria endemic regions (and continually exposed to P. falciparum) may differ significantly from those established in individuals only infrequently exposed to malaria.
In this study, we determined the effect of diminished exposure on the size and function of P. falciparum-specific T cell memory responses by studying a unique epidemiological “natural experiment” on the coast of Kenya. Here, two ethnically and culturally similar cohorts of children with historically endemic exposure to P. falciparum now experience very different levels of exposure. While one group of children has remained continually exposed over the past 8 years (“continually exposed” cohort in Junju), the other has experienced a dramatic reduction in malaria transmission such that exposure has been minimal for over 8 years prior to sampling (“historically exposed” cohort in Ngerenya). By performing a detailed functional characterization of P. falciparum-specific T cell responses found in these two otherwise similar groups of children, we hoped to gain insight into the fate of P. falciparum-specific T cell immunity as exposure to P. falciparum declines. We demonstrate that lack of continuing exposure in the historically exposed cohort resulted in increased levels of P. falciparum-specific CD4+ T cell proliferation and pro-inflammatory cytokine production.
The study took place at the KEMRI-Wellcome Trust Research Programme (KWTRP) situated at the Kilifi County Hospital, Kilifi, Kenya. The hospital serves approximately 500,000 people living in Kilifi County. The children investigated were residents of two villages, located within 20 km of each other, with Junju lying on the southern side and Ngerenya on the northern side of an Indian Ocean creek inhabited predominantly by Mijikenda people.
Characteristics of study cohorts
Mean (95 % CI)
Total no. of previous P. falciparum episodes
Mean (95 % CI)
Time since last episode (mo)
P. falciparum infection status at sampling (% positive)
By blood smear
Sample collection and preparation
Venous blood (5 mL; for immunological studies) and blood smears (for detection and subsequent calculation of P. falciparum parasitemia) were collected from each participant in a preseason cross-sectional survey in May 2012, a time preceded by 4 months of minimal P. falciparum transmission in Junju. PBMCs were isolated by density gradient centrifugation (Ficoll-Histopague, GE Life Sciences) and stored in liquid nitrogen till the assays were performed.
Determination of parasitemia
Thick and thin blood smears were stained with Giemsa and P. falciparum-infected red cells counted against 500 leukocytes and 1000 red blood cells, respectively. To further confirm that previously exposed children were uninfected, a P. falciparum-specific PCR was performed, as previously described .
P. falciparum blood-stage parasites (laboratory-adapted local field isolate) were grown by standard methods and harvested at 5–10 % parasitemia. Red blood cells infected (iRBC) with trophozoite stage parasites were purified via density gradient centrifugation using Percoll (GE, Life Sciences) washed and cryopreserved in glycerolyte. A single batch of parasites was used throughout the study. Aliquots of this batch were stored in liquid nitrogen until required. Uninfected red blood cells (uRBC) were prepared and stored in a similar manner for use as controls.
Intracellular cytokine staining
Thawed PBMCs were rested overnight in media supplemented with 10 % fetal bovine serum (Gibco) and restimulated with media, uRBCs, iRBCs, and plate-bound anti-CD3 (BioLegend) at 7.5 × 105 cells per condition. An effector-to-target ratio of 1:3 was used with uRBCs and iRBCs. Anti-CD28 and anti-CD49d were added for co-stimulation (3 μg/mL BioLegend). Brefeldin A (BD Pharmingen) was added at 6 hours of incubation at a final concentration of 10 μg/mL to inhibit cytokine secretion. At 24 hours, cells were washed, surface stained, fixed, permeabilized, and stained for intracellular cytokines per standard protocols (BD Pharmingen).
Surface and intracellular staining of PBMCs was done with standard staining protocols using the following antibodies: fluorescein isothiocyanate (FITC)-conjugated anti-CD45RO, allophycocyanin (APC)-Cy7-conjugated anti-CD3, Brilliant Violet 421-conjugated anti-IL-10 (panel 1), Brilliant Violet 421-conjugated anti-TNF-α (panel 2), phycoerythrin (PE)-conjugated anti-IL-4 (panel 2), APC-conjugated anti-CCR7 (BioLegend), PerCP-Cy5.5-conjugated anti-CD8, PE-Cy7-conjugated-anti CD27, PE-conjugated anti-IFN-γ (panel 1) (BD Phamingen), and PE-Texas Red-conjugated CD4 (Invitrogen). LIVE/DEAD aqua amine was included to exclude dead cells from downstream analysis (although the malaria specific analysis did not include CD8+ T cells, they were stained to facilitate gating on CD4+CD8– T cells) (see Additional file 1: Table S1 for complete panel).
CFSE proliferation assay
Thawed PBMCs were rested overnight in media supplemented with 10 % fetal bovine serum. Cells were washed and 1 × 106 cells were labeled with 1 mL of 5 μM CFSE (BioLegend) following an established protocol reported elsewhere . CFSE-labeled PBMCs were incubated in a 96-well culture plate and stimulated with media, uRBCs, iRBCs, and plate-bound anti-CD3 (BioLegend) at a density of 2.5 × 105 cells per condition. As before, an effector-to-target ratio of 1:3 was used with uRBCs and iRBCs. Anti-CD28 and anti-CD49d were added for costimulation (3 μg/mL BioLegend). At day 7, supernatants were collected and frozen for downstream cytokine analysis and the cells washed and stained with surface antibodies (Brilliant Violet 421-conjugated anti-CD4, APC-conjugated anti-CCR7, APC-Cy7-conjugated anti-CD3 (BioLegend), PE-Cy7-conjugated anti-CD27, and PerCP-Cy5.5-conjugated anti-CD8 (BD Pharmingen)) before acquisition. Once again, LIVE/DEAD aqua amine was included to exclude dead cells from downstream analysis.
Cytokine production by CD4+ T cells was analyzed using two panels; panel 1: IFN-γ and IL-10; panel 2: IL-4 and TNF-α (both panels assessed surface expression of a number of markers of CD4+ T cell memory phenotype). At least 100,000 lymphocytes were acquired on a 9-color Cyan ADP flow cytometer (Beckman Coulter). Color compensations were performed using samples stained for each of the fluorochromes used. Data were analyzed using FlowJo (Tree Star). Percentages of iRBC-stimulated cytokine producing CD4+ T cells are reported after background subtraction of the frequency of the identically gated population of cells from the same sample stimulated with uRBCs and are expressed as a percentage of total CD4+ T cells. For the phenotypic analysis of CD4 T cell memory subsets, the population of cells that express each marker within the CD4 T cell population was entered into a ‘Boolean gating’ analysis  that separately identifies all the subpopulations expressing each possible combination of markers. The frequency of each specific CD4+ T cell subpopulation is expressed as a percentage of CD4+ T cells. In experiments with CFSE labeled cells, the percentages of divided CD4+ T cells after iRBC stimulation are reported after subtraction of the percentage of divided CD4+ T cells after uRBC stimulation.
Luminex analysis of culture supernatants
Supernatants were thawed and immediately analyzed using a Human Cytokine Magnetic 25-Plex assay (Invitrogen) as recommended by the manufacturer. The following cytokines were measured: IL-1β, IL-1RA, IL-2, IL-2R, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12 (p40), IL-13, IL-15, IL-17, TNF-α, INF-α, IFN-γ, GM-CSF, CCL3 (MIP-1α), CCL4 (MIP1β), CXCL10 (IP-10), CXCL9 (MIG), Eotaxin, CCL5 (RANTES), and CCL2 (MCP-1). Briefly, 50 μL of culture supernatant was diluted 1:2 in medium and incubated with anti-cytokine antibody-coupled magnetic beads for 2 hours at room temperature while shaking at 500 rpm in the dark. The beads were then incubated with 100 μL of a biotinylated detector antibody for 1 hour at room temperature, before incubation with streptavidin R-phycoerythrin for 30 mins (between each step, the beads were washed in wash buffer using a magnetic separator). After a final wash, beads were resuspended in 125 μL of buffer and 100 beads counted for each cytokine in a Bio-Plex MAGPIX multiplex reader (Bio-Rad Laboratories, Inc.). Final concentrations were calculated from the mean fluorescence intensity and expressed in pg/mL using standard curves with known concentrations of each cytokine.
All statistical analyses were performed using Prism 6.0 (GraphPad). Mann–Whitney U-test and Kruskal–Wallis tests were used to compare continuous variables between two and more than two groups, respectively. Correlations between different continuous measures were determined using Spearman’s rank correlation coefficient. For all tests, two-tailed P values were considered significant if P < 0.05.
P. falciparum-specific CD4+ T cells are maintained in the absence of continual exposure
Overall memory phenotype of P. falciparum-specific CD4+ T cells remains unchanged in the absence of continual exposure
P. falciparum-specific T cell proliferation is significantly enhanced after period of minimal exposure
P. falciparum-inducible pro-inflammatory cytokines are upregulated in historically exposed relative to continually exposed children
P. falciparum-specific CD4+ T cell proliferation correlates with recent clinical malaria in continually exposed children
In this study, we sought to determine the effect that declining exposure to P. falciparum has on previously acquired P. falciparum-specific T cell immunity by comparing two cohorts of otherwise similar children with different levels of current exposure. We demonstrate that, while P. falciparum-specific CD4+ T cells are maintained at similar levels and with similar memory phenotype after a significant period (median: 8.7 years; interquartile range: 4.1–9.1 years) of minimal exposure to the parasite, the proliferative capacity of these cells appeared to be altered considerably. In keeping with previous reports [8, 15], we found evidence of significantly impaired CD4+ T cell proliferation in continually exposed children, with levels of P. falciparum-specific CD4+ T cell proliferation in these children indistinguishable from the levels in malaria-naïve children. In contrast, after a period of minimal exposure, P. falciparum-specific CD4+ T cells proliferated robustly upon re-stimulation in vitro, suggesting that the regulatory mechanism responsible for inhibiting P. falciparum-specific CD4+ T cell proliferation is dependent on exposure to the parasite. Importantly, this exposure-dependent inhibition of CD4+ T cell proliferation was restricted to P. falciparum-specific CD4+ T cells, since the levels of CD4+ T cell proliferation in response to anti-CD3 stimulation were similar across the three groups.
While we did not find an association between asymptomatic parasitemia and CD4+ T cell proliferation, we did find a significant positive association between duration since last febrile episode and P. falciparum-specific CD4+ T cell proliferation (in continually exposed children), with more recent febrile episodes associated with lower proliferation. This result is in agreement with a recent study of seasonal malaria in Mali, where successive exposure to P. falciparum resulted in downregulation of pro-inflammatory responses and an upregulation of cytokines responsible for control of inflammation . While that study did not assess P. falciparum-specific CD4+ T cell proliferation, our finding that PBMCs from historically exposed children express higher levels of pro-inflammatory cytokines (including IL-6, IL-5, CXCL9, and CXCL10), but lower levels of anti-inflammatory cytokines (including IL-1RA) than continually exposed children following re-stimulation with P. falciparum suggests that impaired P. falciparum-specific CD4+ T cell proliferation is a further reflection of malaria-induced immunoregulation. Interestingly, IL-6, CXCL9, and CXCL10 have all been demonstrated to stimulate T cell proliferation [50–52], with IL-6 in particular also promoting T cell survival and inhibiting activation-induced cell death . Furthermore, IL-1RA, which we found to be elevated in continually exposed children relative to historically exposed children, has been found to inhibit T cell responses to antigenic stimulation . We acknowledge the findings of a recent study reporting a decline in malaria antigen specific- IFN-γ, IL-10, and TNF-α responses in individuals following a period of low exposure; however, the profile of cytokine responses reported in that study varied substantially by antigen and the reported decline in cytokine levels was not always maintained beyond 6 months of follow-up. Importantly, that study measured cytokine responses to individual peptide antigens, while we measured the response to the whole parasite. The gap in exposure reported was also significantly shorter than in our study, suggesting that, while there might be antigen-specific fluctuations in cytokine levels over short periods of low exposure, the cytokine response profile after longer gaps in exposure may be altered significantly.
Our results provide clear evidence that (1) P. falciparum-specific CD4+ T cells are maintained in the absence of continual exposure to the parasite, (2) continual exposure to P. falciparum induces a strong immunoregulatory response capable of dampening infection-associated inflammation, and (3) P. falciparum-specific CD4+ T cell proliferation (following in vitro stimulation) is significantly enhanced after a period of minimal exposure. While future studies will be needed to precisely define these mechanisms, our data suggests that the mechanisms responsible for mediating malaria-induced immunoregulation (potentially critical for NAI) could be lost in the absence of continual exposure to the parasite. Such mechanisms are likely to involve regulatory T cells [55, 56] and atypical/exhausted lymphocytes [9, 10] that have been shown to expand with continuous exposure to malaria.
As we have mentioned previously, immune individuals who migrate to malaria-free regions for prolonged periods may become susceptible to clinical disease upon re-exposure, but still demonstrate less severe outcomes than malaria-naïve individuals [20–22]. Such individuals mount robust inflammatory responses to relatively few parasites [21, 57], suggesting that they retain some ability to control parasite replication but are unable to modulate malaria-induced inflammation . Considered in the context of our data and existing knowledge about how malaria infection modulates several components of the immune system, these epidemiologic observations suggest a model by which clinical immunity is lost in absence of exposure to P. falciparum. We hypothesize that malaria-naïve individuals who become infected with P. falciparum are unable to control parasite growth resulting in inflammation and associated acute febrile illness. Continually exposed individuals are able to restrict parasite growth (mediated in large part by antibody-dependent mechanisms) and also able to modulate parasite-induced inflammation and T cell proliferation, allowing them to remain afebrile despite the persistence of low level parasitemia. Historically exposed individuals, on the other hand, are capable of limiting parasite growth but have lost the ability to modulate parasite-induced inflammation, resulting in an exaggerated inflammatory reaction and acute febrile illness in response to relatively few parasites. The precise mechanism of malaria-induced immunoregulation remains to be investigated and likely involves both innate and adaptive components. Future work will need to assess the possible contributions of “malaria toxin”-induced TLR hypo-responsiveness , as well as more T cell intrinsic changes that may explain the observed loss of clinical immunity with lack of exposure. One significant caveat of this study is that we are unable to determine whether the immunoregulation observed in historically exposed children represents a decline of pre-established immunoregulatory mechanisms or a failure to adequately develop these mechanisms under conditions of declining exposure. Longitudinal cohort studies and controlled human challenge experiments in naturally immune populations will be needed to fully investigate this question.
The last decade has seen vast investments in malaria control and the associated decline in transmission is reason to be encouraged. However, the heterogeneous nature of this decline  may leave large populations of previously protected individuals susceptible to clinical disease. This is particularly important if a population of children emerge who have lost the ability to control the inflammatory response to malaria and are therefore at higher risk of illness. Furthermore, such pre-existing immune regulation may be responsible for the observed reductions in immunogenicity for malaria experimental vaccines in malaria-exposed populations relative to malaria naïve ones . Understanding the mechanisms of P. falciparum-induced immunoregulation, the role that this plays in NAI, and how immunity is affected by a decline in exposure will be critical in the design and implementation of an effective vaccine, which remains the best long-term preventative measure against malaria.
We thank J. Musyoki for help with P. falciparum culture; O. Kai, D. Kimani, and J. Mwacharo for general assistance; F. Guleid for help with luminex assays; and S. Roetynk, M. Mulongo, F. Osier, and J. Langhorne for useful discussions. Special thanks go to the study participants and their families.
YB is an early career post-doc and for the duration of this project was funded through a Strategic Award awarded to KM and the KEMRI-Wellcome Trust Research Programme.
YB: Conception and design, acquisition of data, analysis and interpretation of data, drafting and revising the article. JMN: acquisition of data, analysis and interpretation of data. GN: contributed unpublished essential data. JW: contributed unpublished essential data. MO: acquisition of data. EWN: conception and design. PB: drafting and revising the article. KM: conception and design. FMN: conception and design, drafting and revising the article. All authors read and approved the final manuscript.
The authors declare that they have no competing interests.
Ethics approval and consent to participate
Informed, written consent was obtained from the parents/guardians of the research participants prior to enrollment in the study. The study was reviewed and approved by the Kenyan Medical Research Institute National Ethics Committee. Results published with the permission of the Director, KEMRI.
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
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