B cell analysis of ethnic groups in Mali with differential susceptibility to malaria

Background Several studies indicate that people of the Fulani ethnic group are less susceptible to malaria compared to those of other ethnic groups living sympatrically in Africa, including the Dogon ethnic group. Although the mechanisms of this protection remain unclear, the Fulani are known to have higher levels of Plasmodium falciparum-specific antibodies of all Ig classes as compared to the Dogon. However, the proportions of B cell subsets in the Fulani and Dogon that may account for differences in the levels of Ig have not been characterized. Methods In this cross-sectional study, venous blood was collected from asymptomatic Fulani (n = 25) and Dogon (n = 25) adults in Mali during the malaria season, and from P. falciparum-naïve adults in the U.S. (n = 8). At the time of the blood collection, P. falciparum infection was detected by blood-smear in 16% of the Fulani and 36% of the Dogon volunteers. Thawed lymphocytes were analysed by flow cytometry to quantify B cell subsets, including immature and naïve B cells; plasma cells; and classical, activated, and atypical memory B cells (MBCs). Results The overall distribution of B cell subsets was similar between Fulani and Dogon adults, although the percentage of activated MBCs was higher in the Fulani group (Fulani: 11.07% [95% CI: 9.317 – 12.82]; Dogon: 8.31% [95% CI: 6.378 – 10.23]; P = 0.016). The percentage of atypical MBCs was similar between Fulani and Dogon adults (Fulani: 28.3% [95% CI: 22.73 – 34.88]; Dogon: 29.3% [95% CI: 25.06 – 33.55], but higher than U.S. adults (U.S.: 3.0% [95% CI: -0.21 - 6.164]; P < 0.001). Plasmodium falciparum infection was associated with a higher percentage of plasma cells among Fulani (Fulani infected: 3.3% [95% CI: 1.788 – 4.744]; Fulani uninfected: 1.71% [95% CI: 1.33 – 2.08]; P = 0.011), but not Dogon adults. Conclusion These data show that the malaria-resistant Fulani have a higher percentage of activated MBCs compared to the Dogon, and that P. falciparum infection is associated with a higher percentage of plasma cells in the Fulani compared to the Dogon, findings that may account for the higher levels of P. falciparum antibodies in the Fulani.


Background
Several studies have demonstrated that individuals of the Fulani ethnic group in West Africa are at lower risk of malaria and tend to have lower Plasmodium falciparum parasite densities compared to individuals of other ethnic groups living sympatrically with the Fulani, including the Dogon [1]. Although protective mechanisms among the Fulani remain unclear, many investigators have consistently shown that the Fulani have higher levels of antibodies specific for P. falciparum antigens expressed at the liver and blood stages [1][2][3][4][5], and enhanced IgG1 and IgG3 subclass and IgM antibody responses to malaria [6]. The B cell biology underlying these observations is not understood.
It is now well established that long-term antibody responses require the generation and maintenance of memory-B cells (MBCs) and long-lived plasma cells (LLPCs), defined in humans by the cell surface markers CD19 + CD27 + CD38 − and CD19 + CD27 ++ CD38 +++ , respectively (reviewed in [7][8][9]). The process of generating MBCs and LLPCs begins when naïve B cells encounter their cognate antigen near the interface of B and T cell areas of secondary lymphoid tissue, which drives naïve B cells to differentiate into isotype-switched short-lived, plasma cells (SLPCs) within the extra-follicular region, which contributes to the initial control of infections. Alternatively, naïve B cells enter follicles where germinal centers are formed, and after a period of 7-10 days, during which the CD4 + T-cell-dependent process of affinity maturation and immunoglobulin class-switching occurs, the germinal center reaction yields LLPCs and MBCs of higher affinity than the initial wave of SLPCs. LLPCs migrate to the bone marrow where they constitutively secrete antibody and provide a critical first line of defense against re-infection, whereas MBCs recirculate and mediate recall antibody responses after re-exposure to their cognate antigen by rapidly proliferating and differentiating into plasma cells.
Recently, it was reported that P. falciparum exposure in Malian children and adults, as well as Peruvian adults [10], is associated with an expansion of a phenotypically distinct population of MBCs identified as CD10 − CD19 + CD20 + CD21 − CD27 − , similar to a MBC subpopulation initially identified in healthy US individuals in mucosalassociated lymphoid tissues by expression of the inhibitory receptor Fc-receptor-like-4 (FCRL4) [11]. B cells with a similar phenotype have been identified in individuals infected with HIV [12] and HCV [13]. Moir et al showed that compared to naïve B cells and classical MBCs, FCRL4 + MBCs proliferated less well in response to BCR-cross-linking and/or to CD40L and Toll-like receptor 9 (TLR9) agonist CpG, and showed a decreased ability to differentiate into antibody secreting cells in response to polyclonal stimulation [12]. FCRL4 + MBCs in HIV-viremic [12] and P. falciparum-exposed individuals [11] also express high levels of inhibitory receptors and a profile of lymphoid-homing receptors similar to that expressed on exhausted CD8 + T cells during chronic viral infections [14]. Given that the function of FCRL4 + MBCs in P. falciparum-exposed individuals is unknown, we refer to this B cell subset in the context of malaria as 'atypical' rather than 'exhausted'.
Here, the proportion of peripheral blood B cells that are immature and naïve B cells; plasma cells; and classical, activated, and atypical MBCs are compared in Fulani and Dogon adults [1], and further comparisons of these B cell subsets in asymptomatic P. falciparum infected and uninfected individuals from both ethnic groups are presented.

Mali study site and participants
This cross-sectional study was done in October 2008 in Mantéourou, Mali, a rural village approximately 850 km north of the capital of Bamako. A detailed description of the study site has been published elsewhere [1]. Participants were randomly selected from an ongoing cohort study which has been described in detail elsewhere [1]. Plasmodium falciparum transmission is seasonal and intense at this site from July through December. The entomological inoculation was approximately 17 infective bites/person/month in September of 2000. This crosssectional study includes 50 adults enrolled in October 2008. As is typical in Mali, the P. falciparum-infected adults had no signs or symptoms of malaria when the blood smear was prepared and they were not treated with anti-malarial drugs (the blood smear was not read immediately). The Ethics Committee of the Faculty of Medicine, Pharmacy, and Odonto-Stomatology at the University of Bamako, Mali approved this study. Deidentified specimens were obtained from this study for immunological analyses. Written, informed consent was obtained from all study participants.

U.S. blood donors
Peripheral blood mononuclear cells (PBMCs) from eight healthy adult blood bank donors in the U.S. were also analysed. Demographic and travel history data were not available from these anonymous donors but prior P. falciparum exposure is unlikely. Blood samples were obtained for research use after written informed consent was obtained from all study participants enrolled in a protocol approved by the Institutional Review Board of the National Institute of Allergy and Infectious Diseases, NIH (protocol # 99-CC-0168).

PBMC isolation, cryopreservation, and recovery
Malian blood samples were drawn by venipuncture into sodium citrate-containing cell preparation tubes (BD, Vacutainer CPT Tubes). PBMCs were isolated according to the manufacturer's instructions and frozen in foetal bovine serum (FBS) (Gibco, Grand Island, NY) containing 7.5% dimethyl sulfoxide (DMSO; Sigma-Aldrich, St. Louis, MO). U.S. blood samples were drawn into heparinized tubes (BD) and PBMC were isolated from whole blood by Ficoll-Hypaque density gradient centrifugation (Amersham Biosciences) according to the manufacturer's instructions, and frozen in same conditions as Malian samples. PBMCs were kept at −80°C for 24 hours using freezing containers (VWR International) to control the rate of freezing and then transferred to liquid nitrogen. For analysis, PBMCs were rapidly thawed in a 37°C water bath, washed first in PBS with 10% heat-inactivated FBS and then in complete RPMI (RPMI 1640 with L-glutamine supplemented with 10% heat-inactivated FBS, penicillin/ streptomycin 10,000 μg/ml, and 50 μM β-mercaptoethanol (all from GIBCO, Invitrogen)).

Measurement of peripheral blood P. falciparum parasitaemia
Thick blood smears were stained with Giemsa and counted against 300 leukocytes. Plasmodium falciparum densities were recorded as the number of asexual parasites/μl of whole blood based on an average leukocyte count of 7,500/μl. Each smear was evaluated separately by two expert microscopists. Any discrepancies were resolved by a third expert microscopist.

Statistical analysis
Data were analysed using GraphPad Prism 5 for Mac OS X (GraphPad Software, version 5.0d). The non-parametric Mann-Whitney test was used to compare continuous variables between groups, and the Fisher's exact test was used to compare categorical variables. For all tests, twotailed p values were considered significant if ≤0.05.

Study subject characteristics
Twenty-five Fulani adults and 25 Dogon adults living in the same village in Mali were included in this study. Demographic and P. falciparum infection status data are shown in Table 1 according to ethnic group. The average age of the Fulani participants was 38.9 years (range: 19-57 years) and 60% were female. The average age of the Dogon participants was 38.0 years (range: 21-57 years) and 36% were female. At the time of the venous blood collection, four Fulani (16%) and nine Dogon (36%) were infected with P. falciparum by bloodsmear. Of those who were infected, the mean parasite densities for the Fulani and Dogon were 2281.25 ± 1285.9 and 2563.5 ± 1652.9 asexual parasites/μl, respectively. All participants were afebrile and asymptomatic at the time of venous blood draw.

Phenotypic analysis of B cell subsets
The proportion of various B cell subsets in the peripheral blood of Fulani (n = 25) and Dogon (n = 25) adults was compared by flow cytometry analysis of thawed PBMCs, which had been collected at the peak of the malaria season. These data were compared to B cell profiles of P. falciparum-naïve U.S. adults (n = 8).
As a percentage of total lymphocytes, the mean percentage of total B cells (CD19 + ) was similar in Fulani and Dogon adults (Figure 2A  Together these data indicate that the overall distribution of B cell subsets is similar among the Fulani and Dogon adults in this study population, with the exception of activated MBCs, which were increased among the Fulani. Activated MBCs are known to promote signaling cascades that induce their differentiation into antibody secreting cells, thus a higher level of activated MBCs among the Fulani is consistent with their propensity toward higher antibody responses and decreased malaria susceptibility [2,6].
The higher percentage of atypical and activated MBCs in Fulani and Dogon Malians versus U.S. adults is consistent with previous studies of P. falciparum-exposed individuals at separate study sites in Mali [11], in The Gambia [15], and Peru [10]. These data provide further evidence that an increase in atypical MBCs is a generalizable finding among P. falciparum-exposed populations. The specificity and function of atypical MBCs in the context of P. falciparum transmission remains to be determined. It is possible that atypical MBCs contribute to protection against malaria by regulating the host immune response; on the other hand, it is also conceivable that functional exhaustion of P. falciparumspecific MBCs through repeated infections contributes to the inefficient acquisition and relatively rapid loss of P. falciparum-specific MBCs and long-lived antibodies which has been observed in areas of intense P. falciparum transmission [16,17].
Also consistent with previous observations [10,11,16], it was found that relative to U.S. volunteers, Malian adults had a lower proportion of immature and naïve B cells and a higher proportion of activated MBCs and plasma cells/plasmablasts, possibly reflecting greater cumulative immunological experience.
To assess whether concurrent asymptomatic P. falciparum infection was associated with alterations in B cell subsets, Fulani and Dogon adults were stratified by whether or not the thick blood smears were positive for P. falciparum infection at the time of blood collection. Plasmodium falciparum infection status was not associated with significant differences in the percentages of total B cells ( Figure 3A). However, P. falciparum infection was associated with a lower percentage of immature B cells among the Fulani ( Figure 3B; Fulani  . The higher percentage of antibody-secreting plasma cells observed in P. falciparum-infected Fulani adults is consistent with the higher levels of P. falciparum-specific antibodies observed by others in this ethnic group [2][3][4]. Asymptomatic P. falciparum infection was not associated with significant changes in the percentages of

Conclusion
It is well established that the Fulani ethnic group is less susceptible to malaria compared to other sympatric ethnic groups in West Africa, but the mechanisms underlying this observation remain obscure. The enhanced P. falciparum-specific antibody response of the Fulani group is among the more consistent observations, which led us to investigate potential differences in B cell subpopulations. Indeed, the Fulani had a higher percentage of activated MBCs compared to the Dogon, and that P. falciparum infection was associated with higher percentage of antibody-secreting cells among the Fulani but not the Dogon. These results serve as a basis for further studies of the B cell biology that underlies the association between Fulani ethnicity and decreased malaria susceptibility. the study in Mali and analysed the clinical data. BT, LHM, MTB, OKD and AD conceived of the study and participated in its design and coordination in Mali. SKP and PDC conceived of the study, participated in its design and coordination in Mali, and helped to analyse the data and draft the manuscript. All authors read and approved the final manuscript.