A novel population of memory‐activated natural killer cells associated with low parasitaemia in Plasmodium falciparum‐exposed sickle‐cell trait children

Abstract Objectives The sickle‐cell trait phenotype is associated with protection from malaria. Multiple molecular mechanisms have been proposed to explain this protection, but the role of the host immune system has been poorly investigated. We hypothesised that cellular immunity to malaria may develop differently in sickle‐cell trait children (HbAS) and children with normal haemoglobin (HbAA) repeatedly exposed to Plasmodium falciparum (Pf). Methods Paired samples collected prior to the Pf transmission season and during the first malaria episode of the ensuing transmission season from HbAS and HbAA children were analysed by multiplex bead‐based assay and comprehensive multi‐dimensional flow cytometry profiling. Results Cellular immune profiles were enriched in HbAS relative to HbAA children before the start of the Pf transmission season, with a distinct NK subset. These cells were identified as a novel subset of memory‐activated NK cells characterised by reduced expression of the ecto‐enzyme CD38 as well as co‐expression of high levels of HLA‐DR and CD45RO. The frequency of this NK subset before the transmission season was negatively correlated with parasite density quantified during the first malaria episode of the ensuing transmission season. Functional assessment revealed that these CD38dim CD45RO+ HLA‐DR+ NK cells represent a important source of IFN‐γ. Conclusion Our data suggest that this novel memory‐activated NK cell subset may contribute to an accelerated and enhanced IFN‐γ‐mediated immune response and to control of parasite density in individuals with the sickle‐cell trait. This distinct cellular immune profile may contribute to predispose HbAS children to a relative protection from malaria.


INTRODUCTION
The sickle-cell trait phenotype results from the heterodimerisation of normal haemoglobin (Hb) A with Hb presenting the b6 Glu ? Val single amino acid substitution known as HbS. Whereas the homodimerisation of mutated HbS is responsible for the sickle-cell disease, which is characterised by a chronic haemolytic anaemia, no severe conditions are associated with the sicklecell trait phenotype (HbAS). On the contrary, it is well established that individuals carrying the heterozygous form HbAS are relatively protected from malaria. 1,2 Specifically, HbAS individuals display a reduced mortality to Plasmodium falciparum (Pf) infection, 3 a reduced parasitaemia during symptomatic malaria 1,4-8 and a delayed onset of malaria, 8,9 as compared with HbAA individuals. Moreover, the frequency of HbAS individuals is particularly high in areas with endemic Pf transmission. 5,10 A number of molecular mechanisms have been proposed to explain the relative protection from malaria displayed by HbAS individuals, including parasite growth inhibition in hypoxic condition, [11][12][13][14] enhanced splenic clearance, 13,15,16 altered cytoadherence, [17][18][19] translocation of HbS-specific parasite growth-inhibiting microRNAs, 20 induction of haem oxygenase-1 21 and HbS polymerisationdependent parasite growth inhibition. 18 A role for the host immune system in the sickle-cell trait phenotype-associated protection has been tentatively proposed, but this has been poorly explored. [22][23][24][25] Few studies have considered the importance of humoral immunity, [26][27][28][29][30] but to the best of our knowledge, there has been no investigation of the role of host cellular immunity in the relative protection of HbAS individuals from malaria.
In malaria-endemic area, naturally acquired immunity is known to develop slowly with age and exposure. [31][32][33] This immunity includes anti-disease or clinical immunity, which protects against disease symptoms, and anti-parasite immunity, which limits blood-stage Plasmodium burden. IFN-c, a key cytokine of the systemic immune response produced by both innate and adaptive immune cells, has been implicated in the protective immunity to Pf infection. [34][35][36][37][38][39] In humans, IFN-c concentration is correlated with protection from symptomatic malaria 40 and resistance to Pf reinfection. 41,42 Ex vivo studies have demonstrated that natural killer (NK) cells are the major source of IFN-c during the very early immune response following Pf infection. 43,44 Although NK cells have been traditionally defined as innate lymphocytes, the existence of memory-like NK subsets, which display an accelerated and enhanced recall response following re-stimulation, has been recently recognised. [45][46][47] In the context of Pf infection, a memory-like status of NK has been suggested based on their adaptive T-cell-dependent IFN-c response 38 and their antibody-dependent cellular toxicity towards parasitised red blood cells. 48 Most recently, in a Malian cohort of Pfexposed individuals, the frequency of an adaptive NK subset has been correlated with protection from malaria. 49 Nonetheless, the IFN-c production and frequency of IFN-c-producing cells, especially of NK cell subsets, have yet to be explored in the context of malaria immunity in HbAS individuals.
In this study, we hypothesised that cellular immunity to malaria may develop differently in HbAS and HbAA children repeatedly exposed to Pf. Accordingly, we compared IFN-c-associated immune profiles of HbAS and HbAA children from a malaria-endemic area of Mali by evaluating (1) the capacity of peripheral blood mononuclear cells (PBMCs) to respond to stimulation, and (2) the frequency of IFN-c-producing cell subsets in total PBMCs, using paired samples collected prior to the Pf transmission season as well as during the first malaria episode of the ensuing transmission season. We then focused our work on NK cells, which were preferentially associated with sicklecell trait-mediated protection, and identified a distinct subset of NK cells that was specifically enriched in HbAS children compared to HbAA children before the start of the Pf transmission season. We defined the phenotypic profile of these NK cells as CD38 dim CD45RO + HLA-DR + and characterised their functional profile.

HbAS children displayed an alteration of their systemic inflammatory response before the start of the Pf transmission season
To investigate potential immune mechanisms involved in the relative protection from malaria associated with the sickle-cell trait phenotype, we first compared the capacity of PBMCs isolated from HbAS and HbAA children repeatedly exposed to Pf to respond to stimulation. PBMCs collected before the start of the Pf transmission season (baseline) and during the first malaria episode of the ensuing transmission season (malaria episode) were stimulated in vitro, and the concentration of a broad range of cytokines and chemokines was measured in supernatants. Of all the cytokines and chemokines tested, only IFN-c was significantly different between HbAS and HbAA children before the start of the Pf transmission season (Figure 1a). PBMCs isolated from HbAS children showed a significantly higher production of IFN-c than HbAA children (median concentration 1494 pg mL À1 vs. 362 pg mL À1 , P < 0.0001; Figure 1b). No significant differences were detected between HbAS and HbAA children during the first malaria episode of the ensuing Pf transmission season for IFN-c or any other cytokine or chemokine (Figure 1c and d).
The frequency of NK cells is increased in HbAS children before the start of the Pf transmission season and correlates with the time to the first malaria episode and with a lower Pf density Since our multiplex screening of cytokine and chemokine production showed that HbAS and HbAA children repeatedly exposed to Pf displayed a significantly different IFN-c response before the start of the transmission season, we next assessed the frequency of PBMC subsets contributing to the systemic IFN-c response. The frequency of CD4 + T cells, CD8 + T cells, cd T cells, NKT and NK cells in HbAS and HbAA children was determined by flow cytometry (Supplementary figure 1). There was no significant difference in the frequencies of CD4 + T cells, CD8 + T cells, cd T cells or NKT cells between HbAS and HbAA children before the start of the Pf transmission season (median frequency 30.1% vs. 26.8%, P = 0.479 for CD4 + T cells; 23.9% vs. 27.1%, P = 0.362 for CD8 + T cells; 3.7% vs. 4.9%, P = 0.266 for cd + T cells; 2.8% vs. 2.6% P = 0.292 for NKT cells; Figure 2a). Surprisingly, NK cells were significantly more frequent in HbAS children than in HbAA children (median frequency 3.8% vs. 2.1%, respectively, P = 0.014, Figure 2a).
We subsequently examined the frequency of NK cells of all available paired samples collected during the first malaria episode of the ensuing transmission season. HbAS and HbAA children displayed similar frequencies of NK cells (median frequency 5.8% vs. 3.5%, P = 0.222; Figure 2b). Because of the unavailability of some HbAS paired samples at the time of the first malaria episode of the ensuing Pf transmission season, statistical analysis of the cell dynamic was compromised. Analysis of available samples is presented in Supplementary figure 2a.
Interestingly, the frequency of NK cells before the start of the Pf transmission season was significantly correlated with the time from the start of the Pf transmission season to the first malaria episode in HbAS children (q = 0.683, P = 0.014; Figure 2c These data demonstrate that HbAS and HbAA children repeatedly exposed to Pf differ in their NK cell profile before the start of the Pf transmission season and suggest that this distinct cellular profile displayed by HbAS children may predispose them to a relative protection from malaria.
The frequency of the CD38 high subset in NK cells before the start of the Pf transmission season correlates with Pf density during the first malaria episode of the ensuing transmission season We have recently reported that the increased expression of CD38, a multifunctional ectoenzyme, on CD4 + and CD8 + T cells is associated with a reduced Pf and Plasmodium vivax density in controlled human malaria infection studies. 50,51 Therefore, we investigated whether significant changes could be detected in CD38 expression on NK cells of HbAS children compared to HbAA children. Among CD38 + cells, we were able to clearly distinguish two cell populations expressing distinct level of CD38: CD38 dim and CD38 high (Supplementary figure 1).
We first analysed the contribution of both CD38 dim and CD38 high subset in the total pool of NK cells. Before the start of the Pf transmission season, HbAS and HbAA children did not display significant difference regarding the blood frequency of the CD38 dim subset in NK cells (median frequency 39.8% vs. 33.9%, P = 0.389; Figure 3a, left panel), whereas the frequency of the CD38 high subset in NK cells of HbAS children was significantly lower in HbAS children (median frequency 37.5% vs. 49.4%, P = 0.027; Figure 3a, right panel). Analysis of the cell dynamic between baseline and the first malaria episode of the ensuing Pf transmission season for available samples is presented in Supplementary figure 2b and c.
No significant correlation was observed between the frequency of either the CD38 dim or the CD38 high subset in NK cells before the start of the Pf transmission season and the time from the start of the Pf transmission season to the first malaria episode neither in HbAS children nor in HbAA children (Supplementary figure 3b and c). The frequency of the CD38 dim subset in NK cells before the start of the Pf was not significantly correlated with the parasite density quantified during the first malaria episode neither in HbAS children (q = À0.034, P = 0.949; Figure 3b, left panel) nor in HbAA children (q = À0.144, P = 0.734; Figure 3b, right panel). Also, the frequency of the CD38 high subset in NK cells and the parasite density were significantly correlated in HbAS children (q = 0.802 P = 0.017; Figure 3c, left panel) but not in HbAA children (q = 0.116, P = 0.784; Figure 3c, right panel), suggesting that the CD38 high fraction of NK cells is not involved in the relative protection from malaria observed in HbAS children repeatedly exposed to Pf. Similar to our previous observation, none of the two CD38expressing subsets in NK cells showed a significant correlation with the axillary temperature neither in HbAS children nor in HbAA children (Supplementary figure 3d and e). Remarkably, in HbAS children, we observed a significant correlation between the frequency of CD38 dim NK cells before the start of the Pf transmission season and the time from the start of the Pf transmission season to the first malaria episode (q = 0.602, P = 0.038; Figure 4b These data demonstrate that the alteration of the NK cell frequency observed before the start of the Pf transmission season in HbAS children repeatedly exposed to Pf is driven by CD38 dim NK cells and suggest a key role for this cell subset in the sickle-cell trait-mediated protection.

CD38 dim NK cells display high IFN-c production and reduced cytotoxic potential
The comprehensive multi-dimensional flow cytometry analysis of HbAS and HbAA children repeatedly exposed to Pf reported above shows that before the start of the Pf transmission season, the frequency of the CD38 high subset in NK cells was reduced ( Figure 3a, right panel), whereas the frequency of CD38 dim NK cells in the total pool of PBMCs was increased (Figure 4a, left panel) in HbAS children compared to HbAA. Thus, we next investigated the functional phenotype of the CD38 dim and CD38 high populations of NK cells. Given the unavailability of additional samples from the HbAS children repeatedly exposed to Pf included in this study, we further expanded our investigation of these interesting cell populations to include uninfected malaria-na€ ıve donors. PBMCs were isolated and then stimulated in vitro,  figure 4) revealed that the CD38 dim and CD38 high NK cell subsets differed in their cytokine production profiles. Specifically, the frequency of IFN-c-producing cells was significantly higher in the CD38 dim NK subset than the CD38 high NK subset (median frequency 29.8% vs. 9.4%, P = 0.031; Figure 5a, left panel), whereas the frequency of TNF-a-producing cells was significantly reduced in the CD38 dim NK subset compared to the CD38 high NK subset (median frequency 37.2% vs. 43.4%, P = 0.003; Figure 5a, middle panel). Examination of polyfunctionality revealed that the CD38 dim NK cells were significantly enriched in cells co-expressing IFN-c and TNF-a compared to their CD38 high counterparts (median frequency 12.7% vs. 3.2%, P = 0.034; Figure 5a, right panel). Moreover, the production of IFN-c as indicated by the mean fluorescence intensity (MFI) was significantly higher in the CD38 dim NK subset than the CD38 high NK subset (median MFI 233.0 vs. 172.5, P = 0.039; Figure 5b, left panel), whereas the production of TNF-a was significantly lower in the CD38 dim NK subset than the CD38 high NK subset (median MFI 255.5 vs. 296.0, P = 0.009; Figure 5b, right panel).
As secretion of cytotoxic granules is also a functional hallmark of NK cells, we next assessed the production of perforin and Granzyme-B by CD38 dim and CD38 high NK cells (Figure 5c and d). Consistent with the observed differences between CD38 dim and CD38 high NK cells with regard to cytokine production, these two subsets also differed in their cytotoxic potential. Specifically, the frequencies of perforin + and Granzyme-B + cells were significantly lower in the CD38 dim NK subset than the CD38 high NK subset (median frequency 48.0% vs. 80.7%, P = 0.007 for perforin + cells; and 53.9% vs. 83.9%, P = 0.015 for Granzyme-B + cells, respectively; Figure 5c, left and middle panel). The CD38 dim NK subset contained a significantly lower proportion of cells coexpressing perforin and Granzyme-B (46.9% vs. 79.5%, P = 0.005; Figure 5c, right panel). Additionally, the production of perforin in the CD38 dim NK subset was approximatively 2.5-fold lower than in the CD38 high NK subset and Granzyme-B production was lower by approximatively 4.5-fold (median frequency 841.5 vs. 2106, P = 0.002 for perforin; and 1201 vs. 4975, P = 0.005 for Granzyme-B, respectively; Figure 5d).
Finally, we determined the contribution of both CD38 dim NK subset and CD38 high NK subset to the production of IFN-c, TNF-a, perforin and Granzyme-B in the total pool of NK cells (Figure 5e). The CD38 dim NK subset was the major source of IFN-c, representing approximatively 65% of the IFN-c-producing NK cell pool. On the contrary, TNF-a, perforin and Granzyme-B production in the total pool of NK cells was largely supported by the CD38 high NK subset, representing approximatively 68%, 89% and 88% of the productive cells, respectively.
The CD38 dim NK subset is enriched in memory-activated cells We next looked in more detail into the phenotype of in vitro stimulated malaria-na€ ıve CD38 dim and CD38 high NK cells. A strong and rapid production of IFN-c and Granzyme-B following Mycobacterium spp. stimulation has been previously linked to the uncommon expression of CD45RO or HLA-DR on NK cells. 45,52 Thus, we hypothesised that the high capacity of CD38 dim NK cells to generate a strong IFN-c response could be associated with expression of memory-and/or activation-associated molecules. Flow cytometry analysis of CD45RO + and HLA-DR + cells (Supplementary figure 5) revealed significant higher frequency of both cell populations in the CD38 dim NK subset than the CD38 high NK subset (median frequency 14.0% vs. 0.65%, P = 0.0005 for CD45RO + cells and 19.0% vs. 4.6%, P = 0.0009 for HLA-DR + cells; Figure 6a). Importantly, these observations were validated in vivo in PBMCs collected before the start of the Pf transmission season from our cohort of Malian individuals repeatedly exposed to Pf (median frequency 4.7% vs. 0.8%, P < 0.0001 for CD45RO + cells and 11.7% vs. 4.3%, P < 0.0001 for HLA-DR + cells; Figure 6b). CD38 dim NK cells were also significantly enriched in cells expressing NKG2C, a NK-specific activating receptor described on adaptive NK cells in the context of CMV infection, 53-55 as demonstrated by in vitro stimulation of malaria-na€ ıve PBMCs (median frequency 18.9% vs. 7.5%, P < 0.0001; Figure 6c and Supplementary figure 6). In both uninfected malaria-na€ ıve donors and Pf-exposed Malian individuals, CD38 dim NK cells retained a CD56 dim phenotype (median frequency 88.2% in malaria-na€ ıve individuals and 82.8% in Pfexposed; Figure 6d).
To further determine to what extent cells expressing memory-or activation-associated molecules were responsible for the cytokine and cytotoxic molecule production detected in the CD38 dim NK cell subset, we determined correlation coefficients between the frequency of CD45ROand HLA-DR-expressing CD38 dim NK cells and the frequency of producing cells, or the production capacity. Importantly, the frequency of the CD45RO + cell subset in CD38 dim NK cells was positively correlated with the frequency of IFN-cproducing cells in CD38 dim NK cells (q = 0.943, P = 0.017; Figure 6e, left panel) and the capacity of CD38 dim NK cells to produce IFN-c (q = 0.847, P = 0.033; Figure 6e, right panel), suggesting that the CD45RO + subset is the main source of IFN-c in CD38 dim NK cells. A similar correlation was observed between HLA-DR + cells in CD38 dim NK cells and IFN-c-producing cells in CD38 dim NK cells (q = 0.943, P = 0.017; Figure 6f, left panel) as well as with their IFN-c production (q = 0.875, P = 0.022; Figure 6f, right panel), suggesting that IFN-c-producing CD45RO + HLA-DR + CD38 dim NK cells might constitute a single distinct subset of NK cells. These observations are consistent with the CD45RO + and HLA-DR + NK subsets being responsible for a robust IFN-c response, as noted previously for Mycobacterium spp. 45,52 Remarkably, CD45RO + cells in CD38 dim NK cells and HLA-DR + cells in CD38 dim NK cells were both negatively correlated with perforin-producing CD38 dim NK cells (q = À0.714, P = 0.136 for CD45RO + cells and q = À0.886, P = 0.033 for HLA-DR + cells; Figure 6g and h, left panel) and perforin production (q = À0.771, P = 0.103 for CD45RO + cells and q = À0.755, P = 0.083 for HLA-DR + cells; Figure 6g and h, right panel).
CD45RO + HLA-DR + CD38 dim NK cells constitute a single distinct subset of NK cells displaying strong IFN-c response following stimulation We next investigated whether CD45RO + cells and HLA-DR + cells constituted two distinct subsets of CD38 dim NK cells or a single subset co-expressing both markers. Flow cytometry analysis revealed that HLA-DR + CD38 dim NK cells were significantly enriched in CD45RO + compared to HLA-DR + CD38 high (median frequency 55.2% vs. 16.1%, P = 0.003; Figure 7a). Similarly, there was a significantly higher frequency of HLA-DR + cells in CD45RO + CD38 dim NK cells than CD45RO + CD38 high NK cells (median frequency 85.5% vs. 73.4%, P = 0.042; Figure 7b). Therefore, the frequency of CD45RO + HLA-DR + double-positive cells was evaluated in CD38 dim and CD38 high NK cells. Our data showed that CD45RO + HLA-DR + cells were significantly more frequent in CD38 dim NK cells than CD38 high NK cells (median frequency 12.5% vs. 0.6%, P = 0.0005; Figure 7c, left panel). Importantly, similar results were also observed in vivo in PBMCs collected before the start of the Pf transmission season from our cohort of Malian individuals repeatedly exposed to Pf (median frequency 1.9% vs. 0.2%, P < 0.0001; Figure 7c, right panel). In accordance with our previous observations, the frequency of CD45RO + HLA-DR + cells in CD38 dim NK cells was significantly correlated with the frequency of IFN-c-producing cells in CD38 dim NK cells (q = 0.817, P = 0.047; Figure 7d, left panel), whereas it was significantly inversely correlated with perforin-producing CD38 dim NK cells (q = À0.886, P = 0.033; Figure 7d, right panel).
Taken together, this study provides the first evidence implicating host cellular immunity in HbAS-associated immune response to Pf infection. We also describe for the first time the existence of a CD38 dim NK subset displaying a memoryactivated phenotype and demonstrate that cellular immune profiles enriched with this distinct NK subset before the start of the Pf transmission season are associated with better parasite control during a subsequent malaria episode. Our data suggest that these memoryactivated CD38 dim NK cells represent an important source of IFN-c production, facilitating parasite control in HbAS individuals.

DISCUSSION
Herein, we report for the first time evidence supporting the existence of a distinct cellular immunity between HbAS and HbAA children repeatedly exposed to Pf. Our work points out the existence of a novel NK cell subset that expresses a reduced level of CD38 and co-expresses high levels of HLA-DR and CD45RO, representing a memory-activated phenotype. Functional assessment revealed that the CD38 dim CD45RO + HLA-DR + NK cells represent an important source of IFN-c. We further showed that these CD38 dim NK cells were specifically enriched in HbAS children compared to HbAA children before the start of the Pf transmission season, and were associated with a longer period between the start of the transmission season and the first malaria episode as well as with a better parasite control during this subsequent malaria episode. Therefore, our study suggests that memoryactivated NK cells present before the start of the Pf transmission season may contribute to control parasites and delay the onset of the next malaria episode in HbAS children by producing an accelerated and enhanced IFN-c response.
There is considerable evidence highlighting the importance of parasite-induced IFN-c responses in the control of Pf malaria, including correlation with protection from symptomatic malaria 40,56 and resistance to Pf reinfection. 41,42 Moreover, the intensity and rapidity of the IFN-c response has been associated with both anti-parasite immunity 57 and clinical immunity. 58 Our work demonstrates for the first time that circulating PBMCs isolated before the start of the transmission season from Pf-exposed HbAS individuals display a higher IFN-c response to stimulation than HbAA individuals. We further dissected the cellular sources responsible for this early IFN-c response. Among all the IFN-cproducing cells explored in our study, we show that NK cells are the only cell subset with a significantly increased frequency in HbAS individuals compared to HbAA before the start of the Pf transmission season. More importantly, the frequency of NK cells present before the start of the transmission season in the peripheral blood of HbAS children was significantly correlated with the time from the start of the Pf transmission season to malaria episode as well as with the capacity to control parasite density.
Although NK cells are often considered as a bulk population of cells expressing CD56, they constitute a highly heterogeneous cellular compartment of the immune system. [59][60][61] Similar to T cells, multiple subsets of NK cells can be distinguished based on the expression of a variety of surface receptors and production of immune mediators, with CD56 bright NK cells being generally considered as relatively immature cells with high cytokine production capacity, whereas CD56 dim NK cells are more mature cells associated with a high cytotoxic activity. 59,62 Heterogeneity of the NK cell response has been demonstrated in Pf infection. 63 In the present study, by assessing the expression level of the ecto-enzyme CD38, we identified a CD38 dim and a CD38 high NK subset. Further analyses demonstrated that CD38 dim and CD38 high NK cells display a heterogeneous capacity to produce cytokines and cytotoxic mediators. Although in our study both CD38 dim and CD38 high NK cell subsets were able to produce IFN-c, TNF-a and cytotoxic granules following stimulation, the CD38 dim NK cell subset preferentially expressed high levels of IFN-c, even though more than 80% of the cells were CD56 dim . Importantly, the CD38 dim NK cell subset was the major source of IFN-c in the total pool of NK cells. Such dichotomy between cytokine production and cytotoxic activity has been previously observed on terminally differentiated CD57 + NK cells in the context of antiviral response. 64 Moreover, even if our analysis did not show any significant difference when comparing the frequency of CD56 dim cells in CD38 high NK cells and CD38 dim NK cells of malaria-na€ ıve individuals (P = 0.266; Supplementary figure 7a), we observed that the frequency of CD56 dim cells was significantly higher in the CD38 high NK subset than CD38 dim NK subset of Pf-exposed children (P < 0.0001; Supplementary figure 7b). Therefore, the relatively lower proportion of CD56 dim cells in the CD38 dim NK cell subset could partially explain their reduced capacity to produce cytotoxic granules. Although, we can not exclude that the lower expression of CD38 may also partly explain the reduced release of Perforin and Granzyme-B by CD38 dim NK cells as signalling through this ecto-enzyme has been previously associated with induction of cytotoxic activity on NK cells. 65,66 Importantly, we demonstrated that HbAS individuals display a significantly higher frequency of CD38 dim NK cells than HbAA individuals before the start of the Pf transmission season. Moreover, we show that the enrichment in CD38 dim NK cells at baseline is associated with both a delay onset of the next malaria episode and a relative control of parasite density during this episode in HbAS individuals. Previously, a blood-stage controlled human malaria infection study by our group associated CD4 + T cells expressing CD38 with reduced Pf density in malaria-na€ ıve volunteers exposed to Pf for the first time 50,51 ; however, NK cells were not investigated in that study. Of note, in our current study, analysis of CD4 + T cells expressing either CD38 dim or CD38 high did not reveal any significant difference between HbAS and HbAA children repeatedly exposed to Pf (Supplementary figure 8).
The expression of CD45RO at the surface of T cells is conventionally used to discriminate their memory and na€ ıve status, whereas the expression of HLA-DR is used to assess their activation status. However, CD45RO and HLA-DR expression on NK cells has been reported. 45,52,67,68 Herein, we were able to detect both CD45RO and HLA-DR on the surface of NK cells in both uninfected malaria-na€ ıve adults and children repeatedly exposed to Pf. We further showed that the CD38 dim NK subset was particularly enriched in both CD45RO + cells and HLA-DR + cells as compared to its CD38 high counterpart, suggesting that CD38 dim NK cells have a memory-activated phenotype. These exciting findings were supported by the high expression of NKG2C, the CD56 dim phenotype and the high cytokine production versus reduced cytotoxic potential observed on CD38 dim NK cells. Similar to memory mechanisms observed in the Tcell compartment, establishment and maintenance of the memory-activated CD38 dim NK cell compartment might constitute in HbAS children an adaptive-like mechanism for optimising the initiation of a very early and strong immune response following Pf re-exposure. In both mice and human, maintenance of memory NK cells has been reported several months post-infection. 69,70 To our knowledge, mechanisms supporting the maintenance of memory NK cells in the circulating blood remain unclear. A distinct chemokine receptor profile might allow memory-activated CD38 dim NK cells to patrol through the circulating blood to the peripheral organs. 71 Also, specific cytokine environment, for example enriched with IL-15, might support the maintenance of such memory NK cells in the circulating blood. 72 Alternatively, we cannot exclude that a subclinical infection (below the detection limit of the Pf-PCR 73 ) might support the maintenance of a memory-activated CD38 dim NK cell compartment in the blood.
Finally, we identified a CD38 dim NK subset coexpressing both CD45RO and HLA-DR. Functionally, the expression of either CD45RO or HLA-DR on NK cells has been previously associated with strong IFN-c and cytotoxic responses following stimulation. 45,52,67 Consistent with those reports, our experiments showed that CD45RO + HLA-DR + CD38 dim NK cells exerted high IFN-c responses following stimulation. However, in contrast to previous studies on CD45RO + NK cells and HLA-DR + NK cells, we observed a reduced cytotoxic response on CD45RO + HLA-DR + CD38 dim NK cells, possibly because of their reduced expression of CD38 65,66 and/or their more mature status. 64 The existence of memory NK cells was first established in rodent models. [74][75][76] The importance of such NK cells has since been highlighted in non-human primates infected with the simianhuman immunodeficiency virus 77 and in human infected by Mycobacterium tuberculosis 45 or CMV 53-55 ; as well as in vaccinated individuals. 46,52,78 Recently, a high frequency of NK dim NKG2C + CD57 + memory-like cells at the time of primary HIV infection was associated with an enhanced early control of the viral load under combined antiretroviral therapy. 79 In the context of Pf infection, the expression of the DNAX accessory molecule 1, a co-stimulatory molecule found essential for the differentiation of memory NK cells, 80 was associated with the NK ability to eliminate parasitised red blood cells. 81 More recently, the frequency of an antibody-dependent memory-like NK cell subset has been correlated with low parasitaemia and resistance to malaria. 49 Herein, by identifying the existence of a novel memory-activated NK cell subset characterised by the CD38 dim CD45RO + HLA-DR + phenotype, we show for the first time the involvement of cellular immunity in the HbAS-associated immune response to Pf infection. Our results show that the enrichment in CD38 dim NK cells before the start of the Pf transmission season plays a key role in the better parasite control observed in HbAS children during the first malaria episode of the ensuing transmission season. Our study has important implications for the understanding of immunity to malaria and the development of effective therapeutic interventions. These data suggest that the induction of an adaptive/memory NK response should be considered in the design of new vaccine candidates against Plasmodium infection.

Subjects
Subjects included in the study presented herein were a subset from individuals aged 3 months to 25 years, enrolled in a previously described cohort study. 82 Briefly, a prospective cohort study was conducted in Kalifabougou, Mali, where intense seasonal Pf transmission occurs during a predictable 6-month period from July through December and where repeated infections are common. 82 At baseline (before the start of the Pf transmission season), all subjects displayed a haemoglobin level > 7 g dL À1 , an axillary temperature ≤ 37.5°C and were free of acute systemic illness and chronic disease. Malaria episodes were detected by passive (self-reporting to local health centre) and active (medical examination and PCR, every 2 weeks from enrolment through December) surveillance. Malaria episodes were defined as parasitaemia ≥ 2500 parasites lL À1 , an axillary temperature > 37.5°C and/or signs and symptoms consistent with malaria. 82 Here, we focus on individuals aged 7-13 years, which is the age range during which the acquisition of clinical immunity to uncomplicated malaria begins in areas of intense malaria transmission. From this subset, we randomly selected 12 HbAA and 12 HbAS children free of Pf infection (as by PCR), with blood samples and clinical data available before the start of the Pf transmission season (Table 1). At the first malaria episode of the ensuing transmission season, paired clinical data were available for 8 HbAA and 6 HbAS individuals (Table 1) and paired blood samples were available for 8 HbAA and 5 HbAS individuals. The four groups were similar in terms of gender ratio (P = 0.551), body weight (P = 0.460), age (P = 0.741) and haemoglobin concentration (P = 0.225) ( Table 1). During the first malaria episode, the axillary temperature was significantly increased in HbAA children (P = 0.003; Table 1) and HbAS children (P = 0.025; Table 1) compared to baseline.

Samples
Venous blood (≤ 8 mL) was collected at baseline and at the time of the first malaria episode into sodium heparin tubes (BD Vacutainer CPT, Franklin Lakes, NJ) and transported to the local laboratory for processing within 3 h of collection. PBMCs were isolated by standard density gradient centrifugation and stored at À80°C overnight before longterm storage in liquid nitrogen.

Haemoglobin type
Haemoglobin typing for HbA, HbS and HbC was determined by high-performance liquid chromatography using a D-10 haemoglobin analyser (Bio-Rad, Hercules, CA).

Parasite density
Parasite density was quantified from genomic DNA of Pf asexual parasites extracted from dried blood spots as previously described. 73 Briefly, the Pf 18S rRNA gene was amplified by 15-cycle standard PCR amplification followed by a qPCR. The limit of detection of the nested qPCR was 0.5 parasites lL À1 .
In vitro functional profile of NK cells Peripheral blood mononuclear cells isolated from malaria-na€ ıve buffy coats were stimulated with 20 ng mL À1 of PMA and 1000 ng mL À1 of Ionomycin (Sigma-Aldrich) for 1 h at 37°C. Monensin A (GolgiStop; BD Biosciences) was added, and cells were incubated for a further 5 h at 37°C. Stimulated cells were then stained with anti-human CD3, CD56, HLA-DR, CD45RO and CD38 mAbs (as described above) for 20 mins; fixed and permeabilised using the Cytofix/ Cytoperm kit (BD Biosciences) following the manufacturer's instructions; and then stained with either anti-human IFN-c-FITC (4S.B3; BD Biosciences) and TNF-a-BV785 (Mab11; BioLegend), or perforin-FITC (B-D48) and Granzyme-B-AF647 (QA16A02) (both BioLegend) mAbs. Functional profile of NK cells was analysed on a BD LSRFortessa X20 driven by FACSDiva software (BD Biosciences). FlowJo software version 10.4 was used for gating.

Statistical analysis
Statistical analyses were performed with Prism 7 (GraphPad Software, Inc., San Diego, CA). Statistical tests used are indicated for each Table and Figure. Normality was assessed by either the d'Agostino-Pearson normality test or the Shapiro-Wilk normality test, if 'n' is too small. P-values < 0.05 were considered statistically significant.

Study approval
The Malian cohort study was conducted in compliance with all applicable federal regulations governing protection of human subjects and was approved by the Ethics Committee of the Faculty of Medicine, Pharmacy and Dentistry at the University of Sciences, Technique and Technology of Bamako and the Institutional Review Board of the National Institute of Allergy and Infectious Diseases, National Institutes of Health. The study is registered on http://www.c linicaltrials.gov (NCT01322581). Written informed consent was obtained from the parents or guardians of participating children. The laboratory study with samples collected during that study was approved by the James Cook University Human Research Ethics Committee (#H7735). Subjects with no reported history of malaria or malaria exposure (malaria-na€ ıve, n = 6) were provided by the Australian Red Cross under a protocol approved by the James Cook University Human Research Ethics Committee (#H6702); written informed consent was obtained from all subjects.