Intrinsic properties of germinal center-derived B cells promote their enhanced class switching to IgE

Abstract

Background: Research on the origins and development of human IgE-expressing (IgE(+) ) cells is required for understanding the pathogenesis of allergy and asthma. These studies have been thwarted by the rarity of IgE(+) cells in vivo and the low frequency of class switch recombination (CSR) to IgE ex vivo. To determine the main source of IgE(+) cells, we investigated the relation between the phenotypic composition of tonsil B cells and the CSR to IgE ex vivo.

Methods: Human tonsil B cells were analyzed by flow cytometry (FACS) and cultured with IL-4 and anti-CD40 to induce CSR to IgE. Naïve, germinal center (GC), early GC (eGC), and memory tonsil B cells were isolated by FACS, and their capacities for IL-4 and anti-CD40 signaling, cell proliferation, and de novo class switching to IgE were analyzed by RT-PCR and FACS.

Results: B cells from different tonsils exhibited varying capacities for CSR to IgE ex vivo. This was correlated with the percentage of eGC B cells in the tonsil at the outset of the culture. Despite relatively poor cell viability, eGC and GC B-cell cultures produced the highest yields of IgE(+) cells compared to naïve and memory B-cell cultures. The main factors accounting for this result were the strength of IL-4R and CD40 signaling and relative rates of cell proliferation.

Conclusions: This study shows that the maturation state of tonsil B cells determines their capacity to undergo class switching to IgE ex vivo, with the GC-derived B cells yielding the highest percentage of IgE(+) cells.

Keywords: IgE class switching; allergy; germinal center; human B cells.

Figure 1

Induction of CSR to IgE is highly variable in tonsil B‐cell cultures. (A) Intracellular staining of IgE and IgG on day 12 of a total B‐cell culture that yielded low levels (low‐switched) and high levels of IgE⁺ (high‐switched) B cells. (B) IgE⁺ and IgG⁺ cell percentages on day 12 of culture. The dotted line on the IgE⁺ cell graph indicates the first quartile (Q1 < 7%; low‐switched) and the third quartile (Q3 > 21%; high‐switched). (C) FACS was used to determine the starting frequencies of each Ig class in tonsil B cells prior to culture. Data represent the mean of percentages ± SD of low‐switched (n = 8) and high‐switched (n = 8) B‐cell cultures.

Figure 2

The starting frequency of eGC and memory B cells is predictive of CSR to IgE. Surface staining of CD27 and CD38 on purified tonsil B cells was used to assess the proportions of naïve, eGC, GC, and memory B cells and plasmablasts at the outset of culture. Representative FACS plots from low‐ and high‐switched cultures are shown in (A), and the data showing the mean (±SD) from low‐switched (n = 8) and high‐switched (n = 7) cell cultures are summarized in (B). (C) Correlation of day 12 IgE⁺ cell percentages with day 0 memory and eGC B‐cell percentages. Correlation analysis was performed using Spearman's rank correlation coefficient (***P < 0.001).

Figure 3

eGC and GC B‐cell cultures yield the highest percentage of IgE⁺ cells. (A) After 7 days in culture with IL‐4 and anti‐CD40, we determined, using a live/dead fixable dead stain kit in combination with the side and forward side scatter gating, the cell viability of the B‐cell cultures. (B) Fold change of MFI of anti‐FAS‐stained cells relative to naïve B cells. (C) FACS dot plots showing the intracellular staining of IgE⁺ and IgG⁺ cells after 7 days of CSR culture. (D) Percentage of IgE⁺ and IgG⁺ cells as determined by FACS analysis of the intracellularly stained day 7 naïve, memory, eGC, GC, and total B‐cell cultures. (E) IgE secretion and IgG secretion on day 7 of the cultures were analyzed by ELISA. Data represent the mean ± standard error of mean (SEM) and show the amounts of secreted IgE and IgG [ng/ml] (*P < 0.05, **P < 0.01, ***P < 0.001).

Figure 4

The yields of IgE⁺ cells in IL‐4 and anti‐CD40 cultures are associated with the proliferative capacity of the cultured cells. (A) Division of cells from a representative low‐switched and high‐switched tonsil B‐cell culture as determined by the CFSE dilution, whereby each peak, as indicated by the numbers, represents successive cell divisions. Data are derived from the results on day 12 of cell culture and are representative of three experiments. (B) Percentage of cells at each divisional peak in the low‐ and high‐switched tonsil B‐cell cultures. Data are derived from the results on day 12 of cell culture and represent the mean of percentages ± SD of low‐switched (n = 3) and high‐switched (n = 3) B‐cell cultures. (C) FACS plots show the proliferative capacity of the sorted naïve and total GC (eGC and GC) B cells, as determined by the CFSE dilution, and the percentage of IgE⁺ cells, as determined by intracellular staining, on day 12 of the IL‐4 and anti‐CD40 cultures. (D) The summarized percentage of proliferating cells in the naïve and eGC/GC B‐cell cultures. Data are derived from the results on day 12 of cell culture and represent the mean of percentages ± SD of naïve (n = 4) and eGC/GC (n = 4) B‐cell cultures. Statistical analysis was performed using an unpaired two‐tailed t‐test (*P < 0.05, **P < 0.01).

Figure 5

Enhanced IL‐4R and CD40 signaling in eGC and GC B cells. (A) IL‐4R and CD40 expression levels on tonsil B cells prior to culture with IL‐4 and anti‐CD40. Data show the percentage change in MFI of anti‐IL‐4R‐ and anti‐CD40‐stained cells relative to naïve B cells. (B) STAT6 and NFκB phosphorylation levels 24 h poststimulation with IL‐4 and anti‐CD40. Data show the fold change in MFI of anti‐p‐STAT6 (Tyr641)‐ and anti‐p‐NFκB p65 (Ser536)‐stained cells relative to naïve B cells. They represent the mean ± SD and are derived from three different tonsils. (C) RT‐PCR analysis of AID, εGLT, and γGLT expression 24 h poststimulation with IL‐4 and anti‐CD40. Data represent the mean ± SD of the relative quantification (RQ) and are derived from three different tonsils. (D) RNA was isolated from the sorted naïve, memory, eGC, and GC B cells on day 0 (preculture) and day 5 of cell culture with IL‐4 and anti‐CD40 antibody, The presence of Iε‐Cμ SCTs (167 bp; direct switching) and Iε‐Cγ SCTs (202 bp; sequential switching) was determined by nested PCR. Because of the nature of the nested PCR, the intensity of the PCR band does not necessarily reflect the amounts of switching occurring in our cultures. The positive control was cDNA from an IL‐4‐ and anti‐CD40‐stimulated tonsil B‐cell culture that yielded high percentages of IgE⁺ cells. The negative control was distilled H₂O. Data are representative of three experiments. M = size marker, and bp = the size of the cDNA in base pairs (*P < 0.05, **P < 0.01, ***P < 0.001).