B Cell Receptor Repertoire Analysis in Malaria-Naive and Malaria-Experienced Individuals Reveals Unique Characteristics of Atypical Memory B Cells

Abstract

Malaria, caused by parasites of the Plasmodium genus, is responsible for significant morbidity and mortality globally. Chronic Plasmodium falciparum exposure affects the B cell compartment, leading to the accumulation of atypical memory B cells (atMBCs). IgM-positive (IgM⁺) and IgG⁺ atMBCs have not been compared in-depth in the context of malaria, nor is it known if atMBCs in malaria-experienced individuals are different from phenotypically similar B cells in individuals with no known history of Plasmodium exposure. To address these questions, we characterized the B cell receptor (BCR) repertoire of naive B cells (NBCs), IgM⁺ and IgG⁺ classical MBCs (cMBCs), and IgM⁺ and IgG⁺ atMBCs from 13 malaria-naive American adults and 7 malaria-experienced Ugandan adults. Our results demonstrate that P. falciparum exposure mainly drives changes in atMBCs. In comparison to malaria-naive adults, the BCR repertoire of Plasmodium-exposed adults showed increased levels of somatic hypermutation in the heavy chain V region in IgM⁺ and IgG⁺ atMBCs, shorter heavy chain complementarity-determining region 3 (HCDR3) in IgG⁺ atMBCs, and increased usage of IGHV3-73 in IgG⁺ cMBCs and both IgM⁺ and IgG⁺ atMBCs. Irrespective of Plasmodium exposure, IgM⁺ atMBCs closely resembled NBCs, while IgG⁺ atMBCs resembled IgG⁺ cMBCs. Physicochemical properties of the HCDR3 seemed to be intrinsic to cell type and independent of malaria experience. The resemblance between atMBCs from Plasmodium-exposed and naive adults suggests similar differentiation pathways regardless of chronic antigen exposure. Moreover, these data demonstrate that IgM⁺ and IgG⁺ atMBCs are distinct populations that should be considered separately in future analyses. IMPORTANCE Malaria, caused by Plasmodium parasites, still contributes to a high global burden of disease, mainly in children under 5 years of age. Chronic and recurrent Plasmodium infections affect the development of B cell memory against the parasite and promote the accumulation of atypical memory B cells (atMBCs), which have an unclear function in the immune response. Understanding where these cells originate from and whether they are beneficial in the immune response to Plasmodium will help inform vaccination development efforts. We found differences in B cell receptor (BCR) properties of atMBCs between malaria-naive and malaria-experienced adults that are suggestive of divergent selection processes, resulting in more somatic hypermutation and differential immunoglobulin heavy chain V (IGHV) gene usage. Despite these differences, atMBCs from malaria-naive and malaria-experienced adults also showed many similarities in BCR characteristics, such as physicochemical properties of the HCDR3 region, suggesting that atMBCs undergo similar differentiation pathways in response to different pathogens. Our study provides new insights into the effects of malaria experience on the B cell compartment and the relationships between atMBCs and other B cell populations.

Keywords: HCDR3; IgM; Plasmodium; adaptive immune response; humoral immunity; somatic hypermutation.

FIG 1

IgM⁺ and IgG⁺ atypical MBCs have different levels of somatic hypermutation. (A) Box plots of the frequency of replacement (R) and silent (S) mutations across the entire V segment in naive B cells (NBCs; n = 13), IgM⁺ classical MBCs (cMBC; n = 20), IgG⁺ cMBCs (n = 20), IgM⁺ atypical MBCs (atMBC; n = 17), and IgG⁺ atMBC (n = 14). Differences between NBCs and most cell other B cell types are statistically significant, and the exact P values are shown in Table S5 at https://doi.org/10.6084/m9.figshare.16449858.v3. US-B, Black U.S. adults; US-W, White U.S. adults; UG, Ugandan adults. (B) Box plots of R and S mutations across the entire V segment comparing malaria-naive (for NBCs, n = 10; for IgM⁺ and IgG⁺ cMBCs, n = 13; for IgM⁺ atMBCs, n = 10; and for IgG⁺ atMBCs, n = 8) and malaria-experienced individuals (for NBCs, n = 3; for IgM⁺ cMBCs, IgG⁺ cMBCs, and IgM⁺ atMBCs, n = 7; and for IgG⁺ atMBCs, n = 6). (C) Box plots of R and S mutations in IGHV4-34 codon 26 (IMGT numbering) in malaria-naive (NBCs, n = 10; IgM⁺ and IgG⁺ cMBCs, n = 13; IgM⁺ atMBCs, n = 10; and IgG⁺ atMBCs, n = 8) and malaria-experienced (NBCs, n = 3; IgM⁺ cMBCs, IgG⁺ cMBCs, and IgM⁺ atMBCs, n = 7; and IgG⁺ atMBC, n = 6) individuals. For all panels, *, P < 0.05; ** P < 0.01; and ***, P < 0.001 as determined by two-way mixed-measures ANOVA with pairwise comparisons using either Tukey’s test (panel A; n = 10) or Šídák's test (panel B and C; n = 5).

FIG 2

HCDR3 physicochemical properties of IgM+ atypical MBCs are similar to those of NBCs. (A) Summary of differences in HCDR3 physicochemical properties in memory B cell subsets in comparison to naive B cells (NBCs). P values are reported in Table S6 at https://doi.org/10.6084/m9.figshare.16449858.v3. (B) Box plots of 8 different HCDR3 physicochemical properties in NBCs (n = 13), IgM⁺ classical MBCs (cMBC; n = 20), IgG⁺ cMBC (n = 20), IgM⁺ atypical MBCs (atMBC; n = 17), and IgG⁺ atMBC (n = 14). *, P < 0.05; **, P < 0.01; ***, P < 0.001 as determined by two-way mixed-measures ANOVA with pairwise comparisons by Tukey’s post hoc test, corrected for multiple comparisons (n = 10). US-B, Black U.S. adults; US-W, White U.S. adults; UG, Ugandan adults.

FIG 3

Malaria-associated IgG⁺ atMBCs have shorter HCDR3 sequences. Box plots comparing HCDR3 length in various B cell subsets between malaria-naive (for NBCs, n = 10; for IgM⁺ and IgG⁺ cMBCs, n = 13; for IgM⁺ atMBCs, n = 10; and for IgG⁺ atMBCs, n = 8) and malaria-experienced (for NBCs, n = 3; for IgM⁺ cMBCs, IgG⁺ cMBCs, and IgM⁺ atMBCs, n = 7; for IgG⁺ atMBCs, n = 14) individuals. *, P < 0.05 as determined by two-way mixed-measures ANOVA with pairwise comparisons using Šídák's test (n = 5).

FIG 4

IGHV gene usage differs by cell type. Volcano plots depicting differences in the expression frequency of IGHV genes between naive B cells (NBCs) and other B cell subtypes. Usage is plotted as the log₂ ratio between IGHV gene usage in IgM⁺ classical MBCs (cMBCs; n = 20), IgG⁺ cMBCs (n = 20), IgM⁺ atypical MBCs (atMBCs; n = 17), or IgG⁺ atMBCs (n = 14) and NBCs (n = 13). Genes with significantly higher usage in the indicated cell type than in NBCs are marked in red, while genes with significantly lower usage are marked in blue. Statistical significance was determined by using a Student's t test with Bonferroni correction for multiple testing (n = 44). Dashed horizontal lines indicate corrected P value of 0.05. Dashed vertical lines indicate log₂ fold change of −0.05 and 0.05. Complete lists of IGHV genes with differential usage in MBC subtypes and their P values are included in Table S10 at https://doi.org/10.6084/m9.figshare.16449858.v3.

FIG 5

P. falciparum exposure influences IGHV gene usage. (A) Volcano plots of IGHV gene usage in IgM⁺ classical MBCs (cMBCs), IgG⁺ cMBCs, IgM⁺ atypical MBCs (atMBCs), and IgG⁺ atMBCs. Usage is plotted as the log₂ of the difference between IGHV gene usage in malaria-experienced (IgM⁺ and IgG⁺ cMBCs, n = 13; IgM⁺ atMBCs, n = 10; and IgG⁺ atMBCs, n = 8) and malaria-naive (IgM⁺ and IgG⁺ cMBCs, n = 7; IgM⁺ atMBCs, n = 7; and IgG⁺ atMBCs, n = 6) donors. Dashed vertical lines indicate cutoff log₂ fold change values of −0.05 and 0.05. Dashed horizontal lines indicate an uncorrected P value of 0.05. When corrected for multiple testing (n = 44), no differences for individual B cell subsets were statistically significant. IGHV3-73 is highlighted in red. (B) Box plots comparing frequency of IGHV3-73 gene expression between malaria-naive (IgM⁺ and IgG⁺ cMBCs, n = 13; IgM⁺ atMBCs, n = 10; and IgG⁺ atMBCs, n = 8) and malaria-experienced (IgM⁺ and IgG⁺ cMBCs, n = 7; IgM⁺ atMBCs, n = 7; and IgG⁺ atMBCs, n = 6) groups in IgG⁺ cMBCs, IgM⁺ atMBCs, and IgG⁺ atMBCs.