FCRL5 Delineates Functionally Impaired Memory B Cells Associated with Plasmodium falciparum Exposure

Abstract

Exposure to Plasmodium falciparum is associated with circulating "atypical" memory B cells (atMBCs), which appear similar to dysfunctional B cells found in HIV-infected individuals. Functional analysis of atMBCs has been limited, with one report suggesting these cells are not dysfunctional but produce protective antibodies. To better understand the function of malaria-associated atMBCs, we performed global transcriptome analysis of these cells, obtained from individuals living in an area of high malaria endemicity in Uganda. Comparison of gene expression data suggested down-modulation of B cell receptor signaling and apoptosis in atMBCs compared to classical MBCs. Additionally, in contrast to previous reports, we found upregulation of Fc receptor-like 5 (FCRL5), but not FCRL4, on atMBCs. Atypical MBCs were poor spontaneous producers of antibody ex vivo, and higher surface expression of FCRL5 defined a distinct subset of atMBCs compromised in its ability to produce antibody upon stimulation. Moreover, higher levels of P. falciparum exposure were associated with increased frequencies of FCRL5+ atMBCs. Together, our findings suggest that FCLR5+ identifies a functionally distinct, and perhaps dysfunctional, subset of MBCs in individuals exposed to P. falciparum.

Fig 1. Whole-transcriptome analysis of atypical and classical MBCs from parasitemic, but asymptomatic, subjects.

Heat map rows represent individual genes, and columns within each MBC grouping represent distinct individuals. Representative genes are depicted based on gene ontology associations with specific functional categories. Average fold difference in expression between atMBCs and classical MBCs pairs is shown, with values in parentheses representing lower expression in atMBCs and all other values representing higher expression in atMBCs. The red and blue heat map is a graphical depiction of the significant differential regulation of each gene in nonclassical memory B cell subsets in the context of HIV infection [18,19,45], CVID [22,23], SLE [21,24,25], HCV infection [26,46], and the tonsil [27,28], as well as previously reported expression in atMBCs in the context of malaria [11,31]. Direction of expression change was assigned based on previously published transcriptome and protein expression profiles as described in the methods, with red representing higher expression in nonclassical subsets, blue representing lower expression, and white representing the lack of any reported change.

Fig 2. Spontaneous IgG secretion by different B cell subsets.

(A) Sorted transitional cells (CD19⁺CD10⁺), CD20⁺ atMBCs (IgG⁺CD21^-CD27^-CD19⁺), classical MBCs (IgG⁺CD21⁺CD27⁺CD19⁺), and CD27^- plasmablasts (CD20^-IgG⁺CD21^-CD27^-CD19⁺) were cultured on anti-IgG ELISpot plates for 18 h without additional stimulation. (B) Gating strategy and frequencies of CD38^hi cells in the above plasmablast gating strategy.

Fig 3. Phenotypic characterization of surface proteins on IgG⁺ atypical MBCs.

(A) Surface expression, expressed as median fluorescence intensity (MFI), of CD85d, CD120b, CD360, CD11c, and IgG (BCR) on IgG⁺ atMBCs and IgG⁺ classical MBCs. Lines between symbols denote MBC subsets from the same subject. Wedges represent means. (B) Labeling of SVT2 mouse fibroblast cell lines that express full-length human FCRL4 or FCRL5 protein by monoclonal antibodies 2A6, 1A3, and 7D11. (C) Labeling of human atMBCs with monoclonal antibodies 2A6, 1A3, and 7D11. (D) Isotype-subtracted MFI of FCRL family member expression (“Net MFI”) on atypical and classical MBCs from highly P. falciparum-exposed individuals. Statistical significance was determined using the Wilcoxon signed-rank test. *, p < 0.05; **, p < 0.01

Fig 4. FCRL5 expression defines a phenotypically distinct subset of IgG⁺ atMBCs.

(A) Representative plot showing heterogeneous expression of FCRL5 on IgG⁺ atypical MBCs. Individual FCRL5⁺ atypical and classical MBC frequencies were determined using gates set with a “fluorescence minus one” control with IgG2b isotype control antibody. (B) Proportion of atypical and classical IgG⁺ MBCs expressing FCRL5. Reported frequencies have been subtracted for isotype-labeled background. (C) Median fluorescence intensity (MFI) of surface markers on FCRL5⁺ vs. FCRL5^- atMBCs and FCRL5⁺ vs. FCRL5^- classical MBCs. Statistical significance was determined using the Wilcoxon signed-rank test. *, p < 0.05; **, p < 0.01; ***, p < 0.001.

Fig 5. Higher exposure to P. falciparum is associated with a higher proportion of atMBCs that express FCRL5.

The proportion of FCRL5⁺ atypical MBCs from individuals living in high exposure (n = 16; Nagongera, Uganda; annual entomologic inoculation rate = 310) vs. moderate exposure (n = 9; Walukuba, Uganda; annual entomologic inoculation rate = 2.8) is shown, p = 0.004. Statistical significance was determined using the Wilcoxon rank-sum test. Multivariate linear regression, including age of subject, yielded similar results.

Fig 6. Recall antibody secretion by different B cell subsets.

Sorted FCRL5⁺ and FCRL5^-, atypical (CD20⁺CD21^-CD27^-IgG⁺) and classical (CD20⁺CD21⁺CD27⁺IgG⁺) MBCs were stimulated for 4 days with CpG, F(ab’)₂ anti-IgG, and autologous T cells. IgG-secreting cells were detected by IgG ELISpot and are reported as the number of IgG secreting cells per 1000 cells sorted on day 0. ASC, antibody-secreting cells. Statistical significance was determined using the Wilcoxon signed-rank test. *, p < 0.05.