A positive correlation between atypical memory B cells and Plasmodium falciparum transmission intensity in cross-sectional studies in Peru and Mali

Abstract

Background: Antibodies that protect against Plasmodium falciparum (Pf) malaria are only acquired after years of repeated infections. The B cell biology that underlies this observation is poorly understood. We previously reported that "atypical" memory B cells are increased in children and adults exposed to intense Pf transmission in Mali, similar to what has been observed in individuals infected with HIV. In this study we examined B cell subsets of Pf -infected adults in Peru and Mali to determine if Pf transmission intensity correlates with atypical memory B cell expansion.

Methodology/principal findings: In this cross-sectional study venous blood was collected from adults in areas of zero (U.S., n = 10), low (Peru, n = 18) and high (Mali, n = 12) Pf transmission. Adults in Peru and Mali were infected with Pf at the time of blood collection. Thawed lymphocytes were analyzed by flow cytometry to quantify B cell subsets, including atypical memory B cells, defined by the cell surface markers CD19(+) CD20(+) CD21(-) CD27(-) CD10(-). In Peru, the mean level of atypical memory B cells, as a percent of total B cells, was higher than U.S. adults (Peru mean: 5.4% [95% CI: 3.61-7.28]; U.S. mean: 1.4% [95% CI: 0.92-1.81]; p<0.0001) but lower than Malian adults (Mali mean 13.1% [95% CI: 10.68-15.57]; p = 0.0001). In Peru, individuals self-reporting ≥1 prior malaria episodes had a higher percentage of atypical memory B cells compared to those reporting no prior episodes (≥1 prior episodes mean: 6.6% [95% CI: 4.09-9.11]; no prior episodes mean: 3.1% [95% CI: 1.52-4.73]; p = 0.028).

Conclusions/significance: Compared to Pf-naive controls, atypical memory B cells were increased in Peruvian adults exposed to low Pf transmission, and further increased in Malian adults exposed to intense Pf transmission. Understanding the origin, function and antigen specificity of atypical memory B cells in the context of Pf infection could contribute to our understanding of naturally-acquired malaria immunity.

Figure 1. Atypical MBCs in individuals from the U.S., Peru and Mali.

Atypical MBCs were detected by flow cytometry and expressed as a percentage of total B cells in the peripheral blood of Pf-naive U.S. adults (n = 10), Pf-infected Peruvian adults (n = 18) and Pf-infected Malian adults (n = 12). Box-and-whisker plots represent the smallest and largest values (whiskers), the lower and upper quartiles (top and bottom of box), and the median (horizontal line across box). The Mann-Whitney test was used to compare continuous variables between groups.

Figure 2. Atypical MBCs in Peruvian adults by self-reported history of prior malaria episodes.

Atypical MBCs were detected by flow cytometry and expressed as a percentage of total B cells in the peripheral blood of Pf-infected Peruvian adults who did (n = 12) or did not (n = 6) self-report a prior history of malaria. Box-and-whisker plots represent the smallest and largest values (whiskers), the lower and upper quartiles (top and bottom of box), and the median (horizontal line across box). The Mann-Whitney test was used to compare continuous variables between groups.

Figure 3. Phenotypic analysis of B cell subsets in individuals from the U.S., Peru and Mali.

The following B cell subsets were quantified by flow cytometry, as detailed in Materials and Methods : plasma cells/blasts (CD19⁺ CD21⁻ CD20⁻), activated MBCs (CD19⁺ CD21⁻ CD27⁺CD20⁺), classical MBCs (CD19⁺ CD27⁺ CD21⁺), atypical MBCs (CD19⁺ CD20⁺ CD21⁻ CD27⁻ CD10⁻) naive B cells and immature B cells (CD19⁺ CD10⁺). B cell subsets are expressed as a percentage of total CD19⁺ B cells in the peripheral blood of Pf-naive U.S. adults (n = 10), Pf-infected Peruvian adults (n = 18) and Pf-infected Malian adults (n = 12). The relative proportions of B cell subsets are shown in stacked plots.

Figure 4. Atypical MBCs and immunoglobulin class switching.

Class-switching of atypical MBCs was assessed by measuring cell surface expression of IgG by flow cytometry in the peripheral blood of Pf-naive U.S. adults (n = 10), Pf-infected Peruvian adults (n = 18) and Pf-infected Malian adults (n = 12). Box-and-whisker plots represent the smallest and largest values (whiskers), the lower and upper quartiles (top and bottom of box), and the median (horizontal line across box). The Mann-Whitney test was used to compare continuous variables between groups.