Alterations in peripheral blood B cell subsets and dynamics of B cell responses during human schistosomiasis

Abstract

Antibody responses are thought to play an important role in control of Schistosoma infections, yet little is known about the phenotype and function of B cells in human schistosomiasis. We set out to characterize B cell subsets and B cell responses to B cell receptor and Toll-like receptor 9 stimulation in Gabonese schoolchildren with Schistosoma haematobium infection. Frequencies of memory B cell (MBC) subsets were increased, whereas naive B cell frequencies were reduced in the schistosome-infected group. At the functional level, isolated B cells from schistosome-infected children showed higher expression of the activation marker CD23 upon stimulation, but lower proliferation and TNF-α production. Importantly, 6-months after 3 rounds of praziquantel treatment, frequencies of naive B cells were increased, MBC frequencies were decreased and with the exception of TNF-α production, B cell responsiveness was restored to what was seen in uninfected children. These data show that S. haematobium infection leads to significant changes in the B cell compartment, both at the phenotypic and functional level.

Figure 1. Serum immunoglobulin analysis.

Serum samples were analyzed for total human IgM, IgG1, IgG2, IgG3, IgG4, IgA by Luminex and IgE by ELISA. Bars represent median with interquartile range. Number of donors in each group: baseline S.h. −ve n = 9, baseline S.h. +ve n = 10, follow-up S.h. −ve n = 8 and follow-up S.h. +ve n = 7.

Figure 2. B cell inflammatory cytokine response, activation and proliferation.

Total peripheral blood B cells were cultured with anti-IgG/IgM (2.5 µg/ml), CpG (5 µg/ml) or anti-IgG/IgM plus CpG for two days, restimulated with PMA/Ionomycin/LPS and BrefA and fixed. Levels of intracellular TNF-α (A), CD23 expression (B) and intracellular Ki-67 (C) were measured in S. haematobium-uninfected children by flow cytometry. Levels of intracellular TNF-α (D), CD23 expression (E) and intracellular Ki-67 (F) following CpG stimulation in infected and uninfected children at baseline and follow-up. Horizontal bars represent median. Number of donors in each group: baseline S.h. −ve n = 10, baseline S.h. +ve n = 9, follow-up S.h. −ve n = 7 and follow-up S.h. +ve n = 7.

Figure 3. MBC analysis.

PBMC were fixed and stained with B cell phenotyping markers (CD19, CD27 and IgD) and analyzed for B cell subsets by flow cytometry. B cell subset analysis was performed as shown in (A) (representative S. haematobium-uninfected child). Proportion of CD19-gated cells that were CD27⁺IgD⁻ (B, switched MBC), CD27⁺IgD⁺ (C, non-switched MBC), CD27⁻IgD⁻ (D, double negative MBC), and CD27⁻IgD⁺ (E, naive B cells) were determined for S. haematobium-infected and uninfected children at baseline and follow-up. (B, C, D, E) Horizontal bars represent median. Number of donors in each group: baseline S.h. −ve n = 19, baseline S.h. +ve n = 21, follow-up S.h. −ve n = 8 and follow-up S.h. +ve n = 7.

Figure 4. Expression of IgM, IgA and IgG on CD27⁺ and DN MBCs.

PBMC were fixed and expression of IgM, IgA and IgG on CD27⁺ B cells was evaluated . Proportions of CD19⁺ gated cells that were IgM⁺IgD⁻CD27⁺ (A), IgA⁺IgM⁻IgD⁻CD27⁺ (B), and IgG⁺IgM⁻IgD⁻CD27⁺ (C) were determined for S. haematobium-infected and uninfected children at baseline. For immunoglobulin expression on DN MBCs, PBMC were fixed and stained with B cell subset markers (CD19, IgD and CD27) and DN MBCs measured for IgM, IgA and IgG expression. Proportion of CD19⁺CD27⁻IgD⁻-gated cells that were IgM⁺ (D), IgA⁺ (E) and IgG⁺ (F) were determined for S. haematobium-infected and uninfected children at baseline. Horizontal bars represent median. Number of donors in each group: S.h. −ve n = 8 and S.h. +ve n = 7.

Figure 5. Atypical MBC analysis.

PBMC were fixed and stained with B cell phenotyping markers (CD19, CD21 and CD27) and analyzed for B cell subsets by flow cytometry. B cell subset analysis was performed as shown in (A) (representative S. haematobium-uninfected child). Proportion of CD19-gated cells that were CD27⁺CD21⁻ (B, activated MBC), CD27⁺CD21⁺ (C, classical MBC), CD27⁻CD21⁻ (D, atypical MBC), and CD27⁻CD21⁺ (E, naive B cells) were determined for S. haematobium-infected and uninfected children at baseline and follow-up. (B, C, D, E) Horizontal bars represent median. Number of donors in each group: baseline S.h. −ve n = 19, baseline S.h. +ve n = 20, follow-up S.h. −ve n = 8 and follow-up S.h. +ve n = 7.

Figure 6. B cell subset inflammatory cytokine response, activation and proliferation.

Total peripheral blood B cells were cultured with anti-IgG/IgM (2.5 µg/ml) for two days, restimulated with PMA/Ionomycin/LPS and BrefA, fixed and stained with B cell subset markers (CD10, CD19, CD21 and CD27) and levels of intracellular TNF-α was measured in S. haematobium-uninfected children by flow cytometry (A). Levels of intracellular TNF-α in activated MBCs (B) and classical MBCs (C) in infected and uninfected children at baseline and follow-up. Horizontal bars represent median. Number of donors in each group: baseline S.h. −ve n = 10, baseline S.h. +ve n = 9, follow-up S.h. −ve n = 7 and follow-up S.h. +ve n = 7.