The Differentiation in vitro of Human Tonsil B Cells With the Phenotypic and Functional Characteristics of T-bet+ Atypical Memory B Cells in Malaria

Abstract

Malaria is a deadly infectious disease associated with fundamental changes in the composition of the memory B cell (MBC) compartment, most notably a large expansion of T-bet⁺ MBCs, termed atypical MBCs. However, we know little about the precursors of atypical MBCs and the conditions that drive their differentiation. We compared the responses of human tonsil naïve B cells, MBCs, and germinal center B cells to a variety of stimulatory conditions. We determined that prolonged antigen presentation in the presence of CpG and IFN-γ induced maximal expression of T-bet and other phenotypic markers of malaria-associated atypical MBCs primarily in naïve B cells in vitro. Importantly T-bet⁺ naïve-derived B cells resembled atypical MBCs in their hypo-responsiveness to signaling through their B cell receptors. Thus, naïve B cells can be induced to differentiate into phenotypically and functionally atypical-like MBCs in vitro under conditions that may prevail in chronic infectious diseases in vivo.

Keywords: B cell receptor signaling; IFN-γ; T-bet; TLR9; atypical memory B cells; malaria.

Figure 1

Tonsil B cells express T-bet upon BCR, TLR9, and IFN-γ stimulation in vitro. Tonsil B cells were cultured in vitro for 40 h with combination of antigen, either soluble or presented on a planar lipid bilayer (PLB) or plasma membrane sheet (PMS), CpG, IFN-γ, or IL-12 + IL-18. T-bet expression in naïve (IgD⁺CD10⁻), memory (IgD⁻CD10⁻), and GC (IgD⁻CD10⁺) B cells was analyzed by flow cytometry. (A) Heat map indicating T-bet expression (gMFI) by naïve, memory, and GC B cells of the three individuals (columns) from three representative experiments. The stimulation conditions are as indicated to the right of heat map and are grouped into single, double, triple, and quadruple stimuli. The columns are subgrouped according to the mode of antigen presentation either attached to PLB or PMS or soluble. The gMFI is calculated for conditions in which a minimum of 5% of the cells were T-bet⁺. (B) Comparison of T-bet expression (gMFI) by naïve, memory, and GC B cells stimulated in vitro with antigen either attached to PLB or PMS or soluble, in the presence of CpG and IFN-γ (n = 4). Data for T-bet expression (gMFI) by naïve, memory, and GC B cells stimulated in vitro on all stimulation conditions is provided in Supplementary Table 1. Data were analyzed using one-way analysis of variance (ANOVA) with Tukey's adjustment. ^*P < 0.05; ^**P < 0.01; ns, not significant. (C) Heat map indicating the percent of T-bet⁺ cells for each of the conditions in (A).

Figure 2

T-bet expression in FACS sorted naïve, memory, and GC B cells upon stimulation in vitro. (A) Tonsil B cells from three individuals were FACS sorted based on IgD and CD10 expression into naïve (CD10⁻IgD⁺), memory (CD10⁻IgD⁻), and GC (CD10⁺IgD⁻) B cells gated as shown (Figure S1B) and cultured in vitro as in Figure 1A with either soluble antigen or antigen presented on PLB in the presence of the stimuli shown. T-bet expression was analyzed by flow cytometry and presented as a heat map indicating T-bet expression (gMFI) (left panel) and the percent of B cells expressing T-bet (right panel). The gMFI was calculated for conditions in which a minimum of 5% of the cells were T-bet⁺. (B) Comparison of T-bet expression (gMFI) by FACS sorted naïve, memory, and GC B cells stimulated in vitro with antigen either attached to PLB or soluble, in the presence of CpG and IFN-γ (n = 3). Data for T-bet expression (gMFI) by FACS sorted naïve, memory, and GC B cells stimulated in vitro on all stimulation conditions is provided in Supplementary Table 1. Data were analyzed using paired t test. ^*P < 0.05.

Figure 3

Tonsil naïve and memory B cells upregulate malaria-associated atypical MBC markers upon BCR, TLR9, and IFN-γ stimulation in vitro. (A) Tonsil B cells were cultured in vitro for 40 h unstimulated or stimulated with antigen presented on PLB in the presence of CpG and IFN-γ. Comparison of the expression of T-bet, FcRL5, CD11c, CD95, CXCR3, and CD86 determined by flow cytometry for naïve (IgD⁺CD10⁻) and memory (IgD⁻CD10⁻) B cells are given as the percent of B cells expressing each marker (n = 7). Data were analyzed using paired t test. ^*P < 0.05; ^**P < 0.01; ^***P < 0.001; ^****P < 0.0001; ns, not significant. (B) Percentage of naïve and memory B cells expressing T-bet, FcRL5, CD11c, CD95, CXCR3, or CD86 either immediately ex vivo or after stimulation in vitro for 40 h under the conditions indicated. Each symbol represents a single individual (n = 3). Black bars indicate the mean value and gray boxes indicate ± 1 s.d. Dotted line indicates mean value for unstimulated cells.

Figure 4

T-bet⁺ cells induced in vitro also express malaria-associated atypical MBC markers. Tonsil B cells were cultured in vitro for 40 h unstimulated or stimulated with antigen on PLB in the presence of CpG and IFN-γ. naïve (IgD⁺CD10⁻) and memory (IgD⁻CD10⁻) B cells were analyzed by flow cytometry for the expression of T-bet and FcRL5, CD11c, CXCR3, CD86, and CD95. (A) Representative flow cytometry plots indicating expression of FcRL5, CD11c, CXCR3, CD86, or CD95 by T-bet⁺ naïve and memory B cells. (B) Comparison of expression of FcRL5, CD11c, CXCR3, CD86, or CD95 by unstimulated or stimulated T-bet⁺ and T-bet⁻ naïve and memory B cells (n = 7). Data for expression of these surface markers by T-bet⁺ naïve and memory B cells stimulated in vitro on all the stimulation conditions is provided in Supplementary Table 2. Data were analyzed using one-way analysis of variance (ANOVA) with Tukey's adjustment. ^*P < 0.05; ^**P < 0.01; ^***P < 0.001; ^****P < 0.0001; ns, not significant.

Figure 5

Tonsil naïve and memory B cells stimulated in vitro upregulate ICOS-L, HLA-DR, CD85j, and CD22 and downregulate CD21 and CD62L. (A) Representative flow cytometry plots of the expression of ICOS-L and HLA-DR by atypical MBCs (CD19⁺ CD21⁻ CD27⁻), classical MBCs (CD19⁺ CD21⁺ CD27⁺), and naïve B cells (CD19⁺ CD21⁺ CD27⁻) in PBMCs isolated from Malian adults with lifelong exposure to malaria. Comparison of expression (gMFI) of ICOS-L and HLA-DR by atypical MBCs, classical MBCs, and naïve B cells (n = 5). Data were analyzed using one-way analysis of variance (ANOVA) with Tukey's adjustment. ^*P < 0.05; ^**P < 0.01; ^***P < 0.001; ^****P < 0.0001; ns, not significant. (B) Representative histograms of tonsil naïve and memory B cells following culture in vitro for 40 h without stimulation (gray shaded area) or stimulated (red tracing) with antigen presented on PLB in the presence of CpG and IFN-γ and analyzed by flow cytometry for the expression of ICOS-L, HLA-DR, CD85j, and CD22. (C) Comparison of the expression (gMFI) of ICOS-L, HLA-DR, CD85j, and CD22 by naïve and memory B cells as in (B) (n = 3). Data were analyzed using paired t test. ^*P < 0.05; ^**P < 0.01. (D) Percentage of naïve and memory B cells expressing CD21 and CD62L after in vitro culture for 40 h either without or with PLB-Ag + CpG + IFN-γ stimulation (n = 7). Data was analyzed using paired t-test. ^*P < 0.05; ns, not significant.

Figure 6

Naïve and memory B cells stimulated in vitro to express T-bet exhibit altered BCR signaling. (A) Schematic representation of the strategy for measuring the response of tonsil B cells stimulated in vitro for 40 h with antigen on PLB in the presence of CpG and INF-γ to subsequent BCR crosslinking. Following in vitro culture B cells were harvested, washed and rested for 1 h at 37°C. Cells were activated with soluble F(ab′)₂ antibodies specific for human IgG + IgA + IgM (H+L) (anti-Ig) for 5 min. Phosphorylation of BCR signaling proteins Syk, BLNK, Igα, and PLCγ2 was analyzed by flow cytometry. (B) Heat map indicating the percent of naïve and memory B cells recovered from in vitro cultures that expressed T-bet and additional malaria-associated atypical MBC markers from five individuals (rows). (C) Comparison of the surface expression of BCR [IgG+IgM+IgA (heavy and light chain)] (gMFI) by naïve and memory B cells recovered from 40 h unstimulated and stimulated cultures (n = 3). Data were analyzed using paired t-test. ns, not significant. (D) Representative histograms indicating expression of phospho-Syk, phospho-BLNK, phospho-PLCγ2, and phospho-Igα in naïve and memory B cells recovered from 40 h cultures in vitro and either activated for 5 min with anti-Ig (blue and red tracings) or left unactivated (shaded gray areas). (E) Comparison of unactivated (empty circles) and anti-Ig activation (solid circles) induced expression (gMFI) of phospho-Syk, phospho-BLNK, phospho-PLCγ2, and phospho-Igα (n = 5) in naïve and memory B cells recovered from 40 h unstimulated and stimulated cultures. Values in the graph indicate the average increase in gMFI induced upon anti-Ig activation [ΔgMFI = gMFI(anti-Ig activated) – gMFI(unactivated)]. Data were analyzed using paired t-test. ^*P < 0.05; ^**P < 0.01; ^***P < 0.001; ns, not significant.