B cell-intrinsic signaling through IL-21 receptor and STAT3 is required for establishing long-lived antibody responses in humans

Abstract

Engagement of cytokine receptors by specific ligands activate Janus kinase-signal transducer and activator of transcription (STAT) signaling pathways. The exact roles of STATs in human lymphocyte behavior remain incompletely defined. Interleukin (IL)-21 activates STAT1 and STAT3 and has emerged as a potent regulator of B cell differentiation. We have studied patients with inactivating mutations in STAT1 or STAT3 to dissect their contribution to B cell function in vivo and in response to IL-21 in vitro. STAT3 mutations dramatically reduced the number of functional, antigen (Ag)-specific memory B cells and abolished the ability of IL-21 to induce naive B cells to differentiate into plasma cells (PCs). This resulted from impaired activation of the molecular machinery required for PC generation. In contrast, STAT1 deficiency had no effect on memory B cell formation in vivo or IL-21-induced immunoglobulin secretion in vitro. Thus, STAT3 plays a critical role in generating effector B cells from naive precursors in humans. STAT3-activating cytokines such as IL-21 thus underpin Ag-specific humoral immune responses and provide a mechanism for the functional antibody deficit in STAT3-deficient patients.

Figure 1.

IL-21 predominantly activates STAT1 and STAT3 in primary human B cells. (A and B) Human splenic B cells were cultured for ~24 h with CD40L plus anti-Ig and then cultured in the absence (red) or presence (blue) of IL-21. Phosphorylation of STAT1, 3, 5, and 6 was determined by intracellular staining. (A) Expression of phospho-STATs after 30 min. (B) Kinetics of IL-21–induced STAT phosphorylation in primary human B cells. These results are representative of two (STAT5 and 6) or three (STAT1 and 3) experiments. (C) Expression of phospho-STATs in EBV-LCLs from normal controls or patients with mutations in STAT3 or STAT1 that were cultured without (red) or with (blue) IL-21 for 30 min. (D) Induction of phospho-STATs in cell lines examined from the indicated number of normal donors (red squares) and STAT3_MUT (black triangles, Src homology 2 domain mutations; blue triangles, DNA-binding domain mutations; green triangles, transactivation domain mutations) and STAT1_MUT patients (orange circles) in response to IL-21. The values represent the fold increase in mean fluorescence intensity of cells stimulated with IL-21 for 30 min over unstimulated cells. The symbols correspond to the mean derived from one to four experiments performed on each cell line; the horizontal bars represent means. The dashed lines indicate a fold change of 1, which represents no change in expression of phospho-STAT proteins in the presence of IL-21. DNABD, DNA-binding domain; MFI, mean fluorescence intensity; SH2, Src homology 2 domain; TA, transactivation domain.

Figure 2.

Mutations in STAT3 severely compromise the generation of memory B cells. (A–C) The frequencies of total B cells (A) or transitional (CD20⁺CD10⁺CD27⁻), naive (CD20⁺CD10⁻CD27⁻), and memory (CD20⁺CD10⁻CD27⁺) B cells (B and C) in healthy donors (HD; n = 26–32), STAT3_MUT patients (n = 10), patients with AD-HIES but WT STAT3 alleles (HIES; n = 4–5), STAT1_MUT patients (n = 5–6), or CVID patients (n = 36) were determined by flow cytometry. The values in B correspond to the frequencies (means ± SEM) of transitional (T), naive (N), and memory (M) B cells in the indicated donor populations. (D) memory (CD20⁺CD10⁻CD27⁺) B cells from healthy donors (HD; n = 32), STAT3_MUT patients (n = 10), WT STAT3 AD-HIES patients (n = 5), and CVID patients (n = 31) were labeled with mAbs specific for IgM, IgG, or IgA. The frequency of memory B cells that were IgM⁺, IgG⁺, or IgA⁺ was then determined. The plots in A and B are from representative healthy donors and patients; the graphs in A, C, and D show data points for all donors and patients examined, and the horizontal bars represent means. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Figure 3.

STAT3 deficiency impairs the generation of Ag-specific memory B cells in vivo. B cells from healthy controls or STAT3_MUT patients were cultured with CD40L, CpG, SAC, IL-10, and IL-21 for 4 d and were recultured for 3 d. The proportion of cells secreting (A) IgM+IgG or (C) antitetanus IgG was determined by ELISPOT. (B) Levels of total Ig (IgM+IgG+IgA) secreted during the secondary culture were determined by ELISAs; the results are expressed as micrograms of Ig secreted per 10⁵ input cells in the secondary culture. Each symbol represents the response from an individual control or patient; the horizontal bars represent means. (D and E) Levels of antitetanus IgG in culture supernatants (Sn; D) or serum (E) from normal controls or STAT3_MUT patients were determined by ELISA. (F) Sera from controls or STAT3_MUT patients (1:40 dilution) were incubated with decreasing amounts of immobilized tetanus toxoid. Binding of antitetanus IgG was determined by ELISA. The results are expressed as the percentage of maximum binding, which is defined as the binding to the highest concentration of tetanus (i.e., 3 μg/ml). In D, the data for the controls represent means ± SEM from eight control donors; each blue line represents data from an individual STAT3_MUT patient (n = 8). The data in E and F represent means ± SEM from four control donors and five STAT3_MUT patients.

Figure 4.

Mutations in STAT3 or STAT1 partially reduce IL-10– and IL-21–mediated proliferation and survival of CD40L-activated B cells. (A and B) Naive B cells were sort purified from healthy controls, STAT3_MUT patients, or a STAT1_MUT patient; labeled with CFSE; and cultured for 5 d with CD40L alone or together with IL-4, IL-10, or IL-21. (A) CFSE profiles of control and STAT3_MUT naive B cells stimulated under the indicated conditions. (B) Frequency of B cells in each division; the values for the control and STAT3_MUT B cells represent means ± SEM from five independent experiments. Data for STAT1_MUT B cells are from a single experiment. (C and D) Naive B cells from healthy controls or STAT3_MUT patients were cultured for 3 d with CD40L alone or together with IL-10 or IL-21. (C) The percentage of apoptotic cells was determined by annexin V staining. The values represent the mean percentage of annexin V⁺ cells. (D) The number of viable cells recovered from each culture was also determined. (E and F) Expression of IL-10R (red; E) and IL-21R (red; F) on naive B cells was determined by flow cytometry. The overlays (blue) represent isotype control mAb. The values are means ± SEM (n = 4) of the fluorescence intensity of cells labeled with the isotype control or anti–IL-10R or –IL-21R mAb.

Figure 5.

Isotype switching, AICDA expression, and SHM are unaffected by mutations in STAT3 or STAT1. Naive B cells from (A) normal controls, (B) STAT3_MUT patients, or (C) a STAT1_MUT patient were labeled with CFSE; cultured for 5 d with CD40L alone or together with IL-4, IL-21, or IL-4/IL-21; and analyzed for division and expression of surface IgG. A–C depict contour plots from one experiment. (D) Frequency of IgG⁺ (±SEM) cells in each division (control and STAT3_MUT: n = 7; STAT1_MUT: n = 1). (E) Naive B cells from healthy controls or STAT3_MUT or STAT1_MUT patients were cultured with CD40L alone or with IL-21. After 5 d, expression of AICDA was determined by quantitative PCR. The results are means ± SEM (control: n = 12; STAT3_MUT: n = 7; or STAT1_MUT: n = 3 experiments) and are expressed relative to GAPDH. (F and G) Naive and memory B cells were isolated from three healthy donors and three STAT3_MUT patients. Ig V_H5 genes were cloned and sequenced. (F) The number of mutations detected in each Ig V_H5 gene isolated from normal and STAT3_MUT naive and memory B cells; the horizontal bars represent means. (G) Distribution of mutations in IgV region genes in control and STAT3_MUT memory B cells.

Figure 6.

STAT3 but not STAT1 is absolutely required for IL-21–induced differentiation of human naive B cells into ISCs. (A–C) Naive B cells from control donors (A) or STAT3_MUT patients (B) were labeled with CFSE and cultured with CD40L alone or together with IL-21. After 5 d, the percentage of CD38^hiCD27^hi cells was determined. (C) Percentage of CD38^hiCD27^hi cells (mean ± SEM; n = 5) generated per division from control and STAT3_MUT naive B cells stimulated with CD40L/IL-21. (D–G) Naive B cells from healthy controls (D–F: n = 12; G: n = 5), STAT3_MUT patients (D–F: n = 7; G: n = 5), or STAT1_MUT patients (n = 4) were cultured with CD40L alone or together with IL-21 (D–F) or CpG (G). The levels of secreted IgM (D and G), IgG (E), and IgA (F) were determined by ELISA after 12 d. Each symbol represents Ig secretion by naive B cells from an individual normal donor or patient; the horizontal bars represent means. (H–K) Naive B cells from healthy controls or STAT3_MUT or STAT1_MUT patients were cultured with CD40L alone (40L) or together with IL-21 (+IL21) for 5 d. Expression of PAX5 (H), PRDM1 (I), XBP1 (J), and BCL6 (K) was determined by quantitative PCR. The results are means ± SEM (control: n = 12; STAT3_MUT: n = 7; or STAT1_MUT: n = 3 experiments).

Figure 7.

Differential requirement for STAT3 in regulating human B cell differentiation. Naive B cells can be induced to undergo proliferation, isotype switching, and differentiation to the PC lineage via distinct pathways: (A) in vitro in response to TD stimulation (CD40L plus IL-21), (B) in vivo in response to TD Ag and subsequent GC formation, and (C) in response to TD and T cell–independent stimuli in the form of CD40L and/or BAFF, APRIL, TLR ligands (e.g., viral double-stranded RNA [TLR3] or CpG [TLR9]), and cytokines. Mutations in STAT3 differentially affect each of these pathways (red text). By impairing the up-regulation of PRDM1 (BLIMP-1) and XBP1, STAT3_MUT B cells are unable to become ISCs in vitro; in contrast, induction of AICDA and Ig isotype switching are intact (A). Although GCs can be detected in STAT3_MUT patients, the output of memory B cells and efficient PCs, as assessed by reduced numbers of CD27+ B cells and serum Ab titers against specific Ag, respectively, is impaired. This may result from reduced induction of BCL6 in response to IL-21. Ig production by STAT3_MUT B cells in response to STAT3-independent TD and T cell–independent pathways is intact; this may explain the normal serum Ig levels in these patients (C).