Atypical memory B cells are greatly expanded in individuals living in a malaria-endemic area

Abstract

Epidemiological observations in malaria endemic areas have long suggested a deficiency in the generation and maintenance of B cell memory to Plasmodium falciparum (Pf) in individuals chronically reinfected with the parasite. Recently, a functionally and phenotypically distinct population of FCRL4(+) hyporesponsive memory B cells (MBCs) was reported to be expanded in HIV-infected individuals with high viral loads. In this study, we provide evidence that a phenotypically similar atypical MBC population is significantly expanded in Pf-exposed Malian adults and children as young as 2 years of age as compared with healthy U.S. adult controls. The number of these atypical MBCs was higher in children with chronic asymptomatic Pf infections compared with uninfected children, suggesting that the chronic presence of the parasite may drive expansion of these distinct MBCs. This is the first description of an atypical MBC phenotype associated with malaria. Understanding the origin and function of these MBCs could be important in informing the design of malaria vaccines.

FIGURE 1

Flow cytometry gating strategies for B cell phenotyping. FACS plots of B cell subsets of a representative malaria naïve US and a Pf-exposed Malian volunteer. Within the CD19⁺ gate the B cell subpopulations are defined as follows: activated memory (orange) (CD27⁺CD21^loCD20^hiCD10^-), classical memory (red) (CD27⁺CD21^hi), atypical memory (blue) (CD27^-CD21^loCD20^hiCD10^-), naïve (green) (CD27^-CD21^hiCD10^-), immature (purple) (CD10⁺) and plasma cells or plasma blasts (pink) (CD27^hiCD21^loCD20^-). A four-color two-stain strategy was used as detailed in Materials and Methods to quantify the number of B cells in each subpopulation.

FIGURE 2

Atypical MBCs are significantly increased in Malian as compared to U.S. volunteers. (A-F) The percent of B cell subsets was determined by flow cytometry with phenotypic analysis of subsets as defined in Fig. 1 and detailed in the Materials and Methods section. B cell subpopulations are expressed as a percent of total CD19⁺ B cells for U.S. adults (n=10), Malian adults (n=14) and Malian children ages 8-10 (n=25) and 2-7 (n=36). The Wilcoxon rank-sum test was used to compare continuous variables between groups. (G) The relative proportions of all the B cell subpopulations as analyzed per total CD19⁺ B cells for each age group are given in stacked plots.

FIGURE 3

The IgG expression of atypical and classical MBCs is similar. Stacked plots showing the proportion of IgG⁺ and IgG^- MBC for both the classical MBC subpopulation and the atypical MBC subpopulation. The phenotyping was as in Fig. 1, detailed in Materials and Methods.

FIGURE 4

Inhibitory and tissue-homing receptor expression is increased and lymph node homing receptor expression is decreased on atypical MBCs relative to classical MBCs. FACS analysis of the expression of inhibitory and homing receptors on naïve B cells (green), atypical MBCs (blue) and classical MBCs (red) on a subset of 12 Malian individuals: 6 children age 2-4 and 6 adults. For each panel (A-F) the top plots show individual MFI values for each cell subpopulation of each individual, as well as the average MFI and standard deviation. Underneath are histograms of the MFI from a representative individual of each subpopulation. The expression of inhibitory receptors (A-D), tissue homing receptors (E-H) and lymph node homing receptors (I-K) is given for classical MBCs, atypical MBCs and naïve B cells (as defined in Fig. 1) using appropriately labeled antibodies specific for CD19, CD27, CD21 and the particular inhibitory and homing receptors indicated. The data for different subsets of B cells from individual donors were paired and the Wilcoxon matched pair test was used for the comparison.

FIGURE 5

The percent of atypical MBCs is larger in children with persistent asymptomatic P.falciparum parasitemia as compared to parasite-free children. Shown is the percent of atypical MBCs per total B cells in children aged 2-10 years (A) with (n=9) or without (n=62) Pf parasitemia at the end of the dry season or (B) with (n=8) or without (n=56) helminth infection. The Wilcoxon rank-sum test was used to compare continuous variables between groups.