Naive and memory human B cells have distinct requirements for STAT3 activation to differentiate into antibody-secreting plasma cells

Abstract

Long-lived antibody memory is mediated by the combined effects of long-lived plasma cells (PCs) and memory B cells generated in response to T cell-dependent antigens (Ags). IL-10 and IL-21 can activate multiple signaling pathways, including STAT1, STAT3, and STAT5; ERK; PI3K/Akt, and potently promote human B cell differentiation. We previously showed that loss-of-function mutations in STAT3, but not STAT1, abrogate IL-10- and IL-21-mediated differentiation of human naive B cells into plasmablasts. We report here that, in contrast to naive B cells, STAT3-deficient memory B cells responded to these STAT3-activating cytokines, differentiating into plasmablasts and secreting high levels of IgM, IgG, and IgA, as well as Ag-specific IgG. This was associated with the induction of the molecular machinery necessary for PC formation. Mutations in IL21R, however, abolished IL-21-induced responses of both naive and memory human B cells and compromised memory B cell formation in vivo. These findings reveal a key role for IL-21R/STAT3 signaling in regulating human B cell function. Furthermore, our results indicate that the threshold of STAT3 activation required for differentiation is lower in memory compared with naive B cells, thereby identifying an intrinsic difference in the mechanism underlying differentiation of naive versus memory B cells.

Figure 1.

Morphology and phenotype of CD27⁺ B cells in STAT3_MUT individuals resemble normal memory B cells. (A–C) PBMCs from normal donors and patients with AD-HIES caused by mutations in STAT3 were stained with mAbs specific for CD20, CD27, and CD23, CD24, CD80, CD86, CD95, or TACI. The forward scatter (FSC) and 90° light/side scatter (SSC) and surface expression of the indicated molecules on CD27⁻ (naive) and CD27⁺ (memory) B cells were determined. The histograms in A and B are from a representative normal donor and patient, respectively, whereas the graphs in C depict the geometric mean fluorescence intensity (MFI) of each of the indicated cellular features for CD27⁻ naive (N) and CD27⁺ memory (M) B cells from 3–12 normal donors and STAT3_MUT patients. Each value represents an individual donor or patient; the horizontal lines correspond to the mean. Because of the large difference in the level of expression of CD23 on normal versus STAT3_MUT naive and memory B cells, individual graphs are depicted for normal and patient B cell subsets. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Figure 2.

IL-21 induces activation of STAT1, STAT3, and STAT5 in human naive and memory B cells. Human naive, IgM memory and isotype-switched memory, or total memory, B cells were sort-purified from normal donor spleens. (A) These B cell subsets were cultured for ∼18 h with anti-Ig, rested, and then cultured in the absence (red histograms) or presence (blue histograms) of IL-21 for 30 min. Phosphorylation of STAT1, STAT3, STAT4, STAT5, and STAT6 was determined by intracellular staining. Histograms on the left show representative staining in naive and memory B cells. Right panels plot increase in mean fluorescence intensity of pSTATs in naive, IgM memory, and isotype-switched memory B cells cultured with IL-21; response of unstimulated cells were normalized to a value of 1.0. These values represent the mean ± SEM of two independent experiments using B cells from different donor spleens. Identical results were obtained when the B cell subsets were prestimulated with CD40L/anti-Ig. (B–D) Human B cell subsets were cultured for ∼18 h with anti-Ig, rested, and then left unstimulated or stimulated with IL-21 or anti-Ig for 15–30 min. Cells lysates were prepared and subjected to SDS-PAGE and Western blotting to detect phosphorylated or total STAT3 (B), phosphorylated or total ERK (C), or phosphorylated AKT or 14.3.3 as a loading control (D). B–D are representative of three to four similar experiments.

Figure 3.

STAT3-deficient memory B cells differentiate into Ab-secreting cells in response to IL-21. (A–D) Naive (CD20⁺CD10⁻CD27⁻IgG⁻) and memory (CD20⁺CD10^-CD27⁺) B cells were sort-purified from normal donors (A and B, n = 16; C and D, n = 7), STAT3_MUT patients (A and B, n = 8; C and D, n = 7), or STAT1_MUT patients (n = 6), and then cultured with CD40L alone or together with IL-21 (A and B) or CpG (C and D). The levels of secreted IgM, IgG, and IgA were determined by ELISA after 10–12 d. The columns represent the mean ± SEM of experiments performed using naive B cells from 7–16 normal donors, 7–8 STAT3_MUT patients, or 6 STAT1_MUT patients. (E and F) Naive and memory B cells were sort-purified from normal donors or STAT3_MUT patients and then cultured with CD40L alone or together with IL-4. Expression of AICDA (E) and Ig ε germline transcript (GLT; F) was determined by qPCR and PCR, respectively. The graphs in E represent the mean ± SEM of three experiments using B cells from different donors or patients. The gel depicted in F is representative of experiments performed using B cells from two to three different donors or patients. (G and H) Naive and memory B cells were sort-purified from a single normal donor or STAT3_MUT patient and then cultured with CD40L alone or together with IL-21 or CpG. Proliferation was assessed after 5 d by determining incorporation of [³H]thymidine during the last 18 h of culture. The graphs are the mean ± SEM of replicate cultures of naive or memory B cells from one normal donor or one STAT3_MUT patient. The annotated values indicate the fold increase in proliferation of normal or STAT3_MUT naive or memory B cells cultured with CD40L/IL-21 or CD40L/CpG over that induced by CD40L alone. (I and J) Normal naive (N) or normal, STAT3_MUT, or STAT1_MUT memory (M) B cells (n = 6; I) or total PBMCs from normal donors or STAT3_MUT patients (n = 4; J) were cultured with CD40L and IL-21 for 10–12 d. The levels of antitetanus IgG in culture supernatants were determined by ELISA using immobilized tetanus toxoid as solid phase Ag. Each symbol represents the response of B cells from an individual control or patient; the horizontal bars represent means. ns, no significant; *, P < 0.05; **, P < 0.01.

Figure 4.

STAT1 mutations do not affect the generation of plasmablasts but impair sustained Ig secretion. (A–F) Naive (A–C) and memory (D–F) B cells were sort-purified from normal donors or STAT1_MUT patients and then cultured (25 × 10³/well/100 µl) with CD40L alone (open symbols in B and E) or together with IL-21 (closed symbols in B and E). The generation of plasmablasts, defined as cells acquiring a CD38^hiCD27^hi phenotype (A, B, D, and E), as well as secretion of IgM, IgG, and IgA (C and F), was determined after 4, 5.5, and 7 d. The contour plots (A and D) are representative of plasmablasts detected after 5 d of culture. The graphs depicting Ig secretion are from cultures of CD40L/IL-21–stimulated B cells. The values represent the mean (±SEM for B, C, E, and F) of experiments using cells from two normal donors and two STAT1_MUT patients. Similar results were obtained in a second independent experiment.

Figure 5.

Induction of the PC transcriptional program is intact in IL-21–stimulated STAT3_MUT memory B cells. (A and B) Naive (CD20⁺CD10⁻CD27⁻IgG⁻; A) and memory (CD20⁺CD10⁻CD27⁺; B) B cells were sort-purified from normal donor controls (Ctl; n = 10 [or 7 for AICDA]), STAT3_MUT patients (n = 5), or STAT1_MUT patients (n = 4) and then cultured with CD40L alone or together with IL-21 (+IL-21) for 5 d. Expression of PAX5, PRDM1, XBP1, IRF4, and AICDA was determined by qPCR. The columns represent the mean ± SEM of experiments performed using naive B cells from 7–10 normal donors, 5 STAT3_MUT patients, or 4 STAT1_MUT patients. Levels of expression are relative to the amount of GAPDH. ns, not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Figure 6.

Memory B cells exhibit greater sensitivity to the differentiation-inducing effects of STAT3-activating cytokines IL-21 and IL-10. (A and B) Naive and memory B cells were sort-purified from normal donor spleens and then cultured with CD40L alone or with increasing concentrations of IL-21 (A) or IL-10 (B). After 4 d, expression of PRDM1 and XBP1 was determined by qPCR. The values are presented as the percentage of the maximum response, defined as the levels of expression induced in memory B cells by the highest dose of IL-21 tested (50 ng/ml). The data represent the mean ± SEM of experiments using naive and memory B cells from three different normal donor spleens.

Figure 7.

Loss-of-function mutation in IL21R abolishes B cell responses to IL-21. (A and B) PBMCs from age-matched normal donors and three patients with loss-of-function mutations in IL21R were labeled with mAb against CD20, CD10, CD27, IgM, IgD, IgG, and IgA. (A) Memory B cells were quantified based on the frequency of CD20⁺ B cells that were CD10⁻CD27⁺. (B and C) The percentages of total B cells in normal donors or IL-21R–deficient patients that coexpressed IgM and IgD (B) and of memory (i.e., CD27⁺) B cells from normal donors and IL-21R_MUT patients that expressed IgD, IgG, or IgA (C) were determined. **, P < 0.01. Each symbol in A and B represents an individual normal donor or IL-21R_MUT patient; the horizontal bars represent means. The values in C represent the mean percentage ± SEM of memory B cells from four normal donors or three IL-21R_MUT patients that express IgD, IgG, or IgA. (D–F) Naive or memory B cells sort-purified from normal donors or IL-21R1_MUT patients were cultured with CD40L alone or CD40L/IL-21 (D and E) or CD40L/IL-4, CD40L/IL-10, or CD40L/IL-21 (F). After 5 d, the percentage of plasmablasts (i.e., CD38^hiCD27^hi) generated (D) and expression of PAX5, AICDA, PRDM1, and XBP1 by cultured naive B cells (F) were determined by flow cytometry or qPCR. The values in D represent the mean (±SEM) percentage of naive B cells that acquired a plasmablast phenotype in response to CD40L/IL-21 in experiments using naive B cells from three different normal donors or one IL-21R_MUT patient. In the absence of IL-21 <0.5% plasmablasts were detected in these cultures. (E) The levels of secreted IgM, IgG, and IgA by naive and memory B cells from normal donors (ND) or IL-21R_MUT patients (Pt) in response to stimulation with CD40L/IL-21 were determined by ELISA after 10 d. The amounts of Ig secreted by IL21R_MUT memory B cells in response to CD40L/IL-21 did not differ from those induced by CD40L alone (not depicted). These data are from one of three experiments using naive B cells from five different healthy controls or two unrelated IL-21R_MUT patients. The values in F represent the mean ± SEM from experiments using two to five different healthy controls or of three experiments using cells from two unrelated IL-21R_MUT patients. The horizontal black lines on the graphs in F indicate a value of 1.0, which corresponds to the relative level of expression of the indicated gene in CD40L-stimulated normal naive B cells.