WO2022022524A1

WO2022022524A1 - Prevention and/or treatment of a disease associated with stat3

Info

Publication number: WO2022022524A1
Application number: PCT/CN2021/108721
Authority: WO
Inventors: Chaohong Liu
Original assignee: Huazhong University Of Science & Technology
Priority date: 2020-07-27
Filing date: 2021-07-27
Publication date: 2022-02-03
Also published as: CN114286684A; CN114286684B

Abstract

Provided are prevention and/or treatment of a disease associated with STAT3 by regulation of 14-3-3 σ.

Description

Prevention and/or Treatment of a Disease Associated with STAT3

Technical Field

The present application is directed to prevention and/or treatment of a disease associated with STAT3 by regulation of 14-3-3σ.

Background

Signal transducer and activator of transcription 3 (STAT3) is a member of the STAT protein family, which consists of transcription factors that regulate genes involved in many cellular functions. STAT proteins areactivated by Janus kinases (JAK) . Following stimulation by cytokines or growth factors, membrane receptor-associated JAK phosphorylate STAT3, which then forms homo-or heterodimers to translocate into the nucleus and perform transcription. Because STAT3 controls the expression of a variety of genes and plays a critical role in many cellular processes, including cell growth and apoptosis ¹, STAT3 KO is embryonic lethal in mouse models ².

The function of STAT3 has been studied extensively in immune cells. STAT3 is indispensable for the differentiation of TH17 cells ^3-6. Mice with STAT3 deficiency in T-cells lose the full ability to produce T-follicular helper (Tfh) cells and antibodies during viral infection ⁷. The notable STAT3 mutation in humans is the loss-of-function (LOF) mutation, which leads to hyperimmunoglobulin E syndrome (HIES) . Patients with this disease have recurring infections as well as developmental abnormalities of the bones and teeth ⁸ _, ⁹. On the other hand, gain-of-function mutations (GOF) in the STAT3 gene cause autoimmune diseases ¹⁰. Mutations in other genes such as Dock8, PGM3, SPINK5, and TYK2 have also been reported to induce HIES ^11-14. We generated DOCK8 mutant mice to mimic the mutation in DOCK8 patients and found that the early activation of memory B cells was disrupted. Additionally, deficiency of DOCK8 leads to reduced CD19 and WASP expression ¹⁵. Previous research has shown that STAT3 is downstream of DOCK8 in TLR9-mediated B cell activation and that DOCK8 inhibits IgE production via activating STAT3 ¹⁶. However, whether the correlation between STAT3 and DOCK8 to regulate IgE production exists or not is unknown.

The link between microRNA (miRNA) and hyper-IgE has been recently uncovered. By using miRNA146A transgenic mice, it has been demonstrated that miRNA146A promotes IgE class switching by upregulating 14-3-3σ, a critical factor for immunoglobulin class-switch DNA recombination (CSR) in B cells ^17-19. Ectopic expression of miRNA146A in Jurkat T cells results in moderate down-regulation of STAT3 expression ²⁰. Additionally, the expression of miRNA146A is reduced when the activation of STAT3 is blocked in hepatocellular carcinoma (HCC) cells ²¹. Furthermore, miRNA146A controls SOCS1/STAT3 and cytokine expression in monocytes during hepatitis C infection ²². However, the underlying mechanism of HIES caused by STAT3 mutation, especially in B cells, is still unclear.

B cell receptor (BCR) signaling is vital in regulating B cell processes and functions. BCR signaling decides the fate of B cells and the degree of their antibody response. When antigens bind to BCRs, it causes a conformational change in the receptor and triggers a signaling cascade, such as phosphorylation of CD19, Lyn, Syk, Btk, PLCγ, as well as the negative regulators in BCR signaling, including SHIP and PTEN ^23-25. BCR signaling also induces actin reorganization via several actin regulators, such as WASP, abp1, and N-WASP, which, in turn, gives feedback to BCR signaling via modulating the movement of BCRs ^26-29. How STAT3 affects BCR signaling is unknown in STAT3 LOF and GOF patient B cells.

In one study, STAT3 deficiency blocked the early development of bone marrow B cells, but the deletion of stat3 was driven under Mx1 ^Cre, which is not B cell specific ³⁰. In contrast, no developmental defect was observed in CD19 ^Cre Stat3 ^fl/flmice, whereby the deletion of stat3 was during the later stages of B cell development, and only the T cell-dependent (TD) IgG response was profoundly impaired and not the TD IgM, IgE, and IgA responses or the T cell-independent (TI) IgM and IgG3 responses ³⁰ _, ³¹. STAT3 signaling is essential for the maintenance of the germinal center (GC) and accounts for the pathogenesis of systemic lupus erythematosus (SLE) by regulating GC activities ³². Memory B cells including immunoglobulin switched and unswitched are reduced in STAT3 patients ^33-35. In order to precisely investigate the underlying mechanism of HIES caused by STAT3 mutation in only B cells using a mouse model, we generated Mb1 ^Cre stat3 ^flox/flox mice (STAT3 KO) that have stat3 deleted in the early stages of B cell development.

Summary of the Present Application

In the first aspect, the present application provides a method for prevention and/or treatment of a disease associated with STAT3 in a patient in need of such prevention and/or treatment, comprising regulation of 14-3-3σ in the patient.

In an embodiment of the first aspect, the STAT3 is STAT3 with loss of function or STAT3 with gain of function. In another embodiment of the first aspect, the STAT3 is STAT3 with loss of function.

In another embodiment of the first aspect, the disease associated with STAT3 is hyper-IgE syndrome.

In another embodiment of the first aspect, the regulation of 14-3-3σ is down-regulation of 14-3-3σ or up-regulation of 14-3-3σ. In another embodiment of the first aspect, the regulation is down-regulation of 14-3-3σ. In another embodiment of the first aspect, the down-regulation of 14-3-3σ is conducted with an inhibitor of 14-3-3σ. In another embodiment of the first aspect, the inhibitor of 14-3-3σ is a peptide. In another embodiment of the first aspect, the peptide comprises the amino acid sequence NH ₂-YGRKKKRQRRRPHCVPRDLSWLDLEANMCLP-COOH.

In the second aspect, the present application provides a regulator of 14-3-3σ, a pharmaceutical composition comprising the regulator of 14-3-3σ or a kit comprising the regulator of 14-3-3σ, for use in prevention and/or treatment of a disease associated with STAT3 in a patient in need of such prevention and/or treatment.

In an embodiment of the second aspect, the regulator of 14-3-3σ is a down-regulator of 14-3-3σ or an up-regulator of 14-3-3σ. In another embodiment of the second aspect, the regulator of 14-3-3σ is a down-regulator of 14-3-3σ. In another embodiment of the second aspect, the down-regulator of 14-3-3σ is an inhibitor of 14-3-3σ. In another embodiment of the second aspect, the inhibitor of 14-3-3σ is a peptide. In another embodiment of the second aspect, the peptide comprises the amino acid sequence NH ₂-YGRKKKRQRRRPHCVPRDLSWLDLEANMCLP-COOH.

In another embodiment of the second aspect, the STAT3 is STAT3 with loss of function or STAT3 with gain of function. In another embodiment of the second aspect, the STAT3 is STAT3 with loss of function.

In another embodiment of the second aspect, the disease associated with STAT3 is hyper-IgE syndrome.

In another embodiment of the second aspect, the pharmaceutical composition further comprises a pharmaceutically acceptable excipient.

In another embodiment of the second aspect, the regulator of 14-3-3σ comprised in the kit is in the form of a pharmaceutical composition. In another embodiment of the second aspect, the kit further comprises an instruction for applying the regulator of 14-3-3σ.

In the third aspect, the present application provides use of a regulator of 14-3-3σ in prevention and/or treatment of a disease associated with STAT3 in a patient in need of such prevention and/or treatment.

In an embodiment of the third aspect, the regulator of 14-3-3σ is a down-regulator of 14-3-3σ or an up-regulator of 14-3-3σ. In another embodiment of the third aspect, the regulator of 14-3-3σ is a down-regulator of 14-3-3σ. In another embodiment of the third aspect, the down-regulator of 14-3-3σ is an inhibitor of 14-3-3σ. In another embodiment of the third aspect, the inhibitor of 14-3-3σ is a peptide. In another embodiment of the third aspect, the peptide comprises the amino acid sequence NH ₂-YGRKKKRQRRRPHCVPRDLSWLDLEANMCLP-COOH.

In another embodiment of the third aspect, the STAT3 is STAT3 with loss of function or STAT3 with gain of function. In another embodiment of the third aspect, the STAT3 is STAT3 with loss of function.

In another embodiment of the third aspect, the disease associated with STAT3 is hyper-IgE syndrome.

In the fourth aspect, the present application provides use of a regulator of 14-3-3σ in manufacture of a medicament for prevention and/or treatment of a disease associated with STAT3 in a patient in need of such prevention and/or treatment.

In an embodiment of the fourth aspect, the regulator of 14-3-3σ is a down-regulator of 14-3-3σ or an up-regulator of 14-3-3σ. In another embodiment of the fourth aspect, the regulator of 14-3-3σ is a down-regulator of 14-3-3σ. In another embodiment of the fourth aspect, the down-regulator of 14-3-3σ is an inhibitor of 14-3-3σ. In another embodiment of the fourth aspect, the inhibitor of 14-3-3σ is a peptide. In another embodiment of the fourth aspect, the peptide comprises the amino acid sequence NH ₂-YGRKKKRQRRRPHCVPRDLSWLDLEANMCLP-COOH.

In another embodiment of the fourth aspect, the STAT3 is STAT3 with loss of function or STAT3 with gain of function. In another embodiment of the fourth aspect, the STAT3 is STAT3 with loss of function.

In another embodiment of the fourth aspect, the disease associated with STAT3 is hyper-IgE syndrome.

Summary of the Figures

Figure1: STAT3 is essential for maintaining the homeostasis of peripheral B cells, but not for bone marrow B cells.

(A-C) Flow cytometry data of pre-pro-B cells (A) , pro-B cells (B) , early pre-B cells (C) , late pre-B cells (D) , immature B cells (E) , and recirculating B cells (F) from bone marrow cells of WT and STAT3 KO mice (n=8) , and their respective percentages and absolute numbers. (D) The MFI of CD127 in different populations gated from A and B. (E-H) Flow cytometry data of T1, T2, FO cell populations from WT and STAT3 KO mouse splenic cells (n=8) and their respective percentages and absolute numbers. (I and J) Flow cytometry data of MZ B cells from mouse splenic cells (n=8) and their percentages and absolute numbers. (K and L) Flow cytometry data of GC cells and their percentages and absolute numbers from mouse splenic cells (n=8) . (M-O) Flow cytometry analysis of B1a and B1b cells from WT and STAT3 KO mice (n=6) and their respective percentages and absolute numbers. (P-R) Flow cytometry data of IgE ⁺ splenic B cells from WT and STAT3 KO mice with or without stimulation of anti-CD40 and IL-4 for 5 days (n=4) and their percentages and absolute numbers. (S) Titer of IgE from WT and STAT3 KO mice serum by ELISA (n=6) . Shown are the representative results from three independent experiments. *P <0 . 05 and **P <0. 01.

Figure2: STAT3 positively regulates the proximal BCR signaling.

(A and B) Splenic B cell from WT and STAT3 KO mice were labeled and stimulated with soluble antigen (sAg) using AF546-monobiotinylated-Fab’-anti-IgG plus streptavidin at 37℃ for varying lengths of time. After fixation and permeabilization, the cells were stained for pCD19, imaged by confocal microscopy, and the correlation coefficient between the BCR and pCD19 was quantified. (C) pCD19/CD19 levels in splenic B cells from WT and STAT3 KO mice stimulated with sAg and analyzed by Western blot. (D and E) Confocal microscopy images of pY and pBtk in mouse splenic B cells stimulated with sAg and quantified for correlation coefficient between BCR and pY/pBtk. (F) pY and pBtk/Btk levels in mouse splenic B cells stimulated with sAg and analyzed by Western blot. (G and H) Confocal microscopy analysis of pSHIP in mouse splenic B cells stimulated with sAg and measured for correlation coefficient between BCR and pSHIP. (I) Western blot of pSHIP/SHIP levels in mouse splenic B cells stimulated with sAg. Shown are the representative results from three independent experiments. Scale bar, 2.5 μm. Correlation coefficients were quantified for more than 50 cells using NIS-Elements AR 3.2 software. Mann-Whitney U test (Fig 2 B, E, H) . *P＜0.05; **P＜0.01.

Figure3: The deficiency of STAT3 reduces the accumulation of F-actin mediated by WASP and WIP as well as BCR clustering and positive signalosome recruitment.

(A) Confocal microscopy analysis of pWASP and actin in splenic B cells from WT and STAT3 KO mice. B cells were stimulated with sAg at 37℃ for varying lengths of timeand then fixed, permeabilized, and stained for pWASP and actin using phalloidin. (B) Splenic B cells from WT and STAT3 KO mice were labeled with BV510-anti-B220, stimulated with sAgs at 37℃ for varying lengths of time, and then fixed, permeabilized and stained as in A. The MFI of pWASP and actin were measured by flow cytometry. (C) pWASP/WASP levels in splenic B cells from WT and STAT3 KO mice stimulated with sAg and analyzed by Western blot. (D) Expression of WIP and DOCK8 in splenic B cells from WT and STAT3 KO mice (left panel) and healthy control (HC) and loss-of-function (LOF) STAT3 patient’s PBMC (right panel) . GAPDH was used as a loading control. (E-J) Splenic B cells from WT and STAT3 KO mice were stimulated on membrane-tethered antigen (mAg) using AF546-monobiotinylated-Fab’-anti-IgG for varying lengths of time, then fixed, permeabilized and stained for pWASP and actin. Cells were analyzed using TIRFm and the MFI of the BCR, pWASP, and actin were measured, along with the B cell contact area using the interference reflection microscopy (IRM) . Shown are representative images and the average mean values (±SD) in which more than 50 cells were analyzed using NIS-Elements AR 3.2 software. Scale bars, 2.5 mm. *P＜0.05; **P＜0.01. Mann-Whitney U test (Fig 2G-J) .

Figure4: Deficiency of STAT3 leads to decreased recruitment of pCD19, pBtk and pY, but increased recruitment of pSHIP.

(A and D) Splenic B cells from WT and STAT3 KO mice were stimulated with mAg at 37℃ for varying lengths of time, then fixed, permeabilized and stained for pCD19. Images were taken using TIRFm and the MFI of pCD19 in the B cell contact zone was analyzed. (B, E, F) Splenic mouse B cells stimulated with mAg and stained for pBtk and pY were imaged using TIRFm and the MFI of pBtk and pY in the contact zone were measured. (C and G) Splenic mouse B cells stimulated with mAg and stained for pSHIP were imaged with TIRFm and the MFI of pSHIP in the contact zone was measured. Shown are representative images and the average mean values (±SD) in which more than 50 cells were analyzed using NIS-Elements AR 3.2 software from 3 independent experiments. Scale bars, 2.5 mm. *P＜ 0.05; **P＜0.01. Mann-Whitney U test (Fig 2D, E, F, G) .

Figure5: STAT3 deficiency reduces the PI3K-Akt-mTORC1 mediated metabolic signaling pathway.

(A) Splenic B cells from WT and STAT3 KO mice were stimulated with sAg for varying times, lysed and probed for pPI3k/PI3k, pAkt/Akt, pFoxo1/Foxo1, pMtor/Mtor, and pS6/S6 using Western blot. (B) The basal oxygen consumption rate (OCR) of splenic B cells from WT and STAT3 KO mice responding to treatment of inhibitors oligomycin, FCCP (carbonyl cyanide p-trifluoromethoxyphenylhydrazone) and rotenone. (C and D) Splenic B cells (1x10 ⁶) from WT and STAT3 KO mice (n=4) were labeled with Cell Trace Violet and stimulated with LPS and CpG for 72 hours. The proliferation of B220 ⁺ cells were examined by flow cytometry and the percentage of proliferating cells was quantified. (E and F) The expression of Annexin V from splenic mouse B220 ⁺ cells after stimulation with LPS and CpG for 72 hours. The percentage of Annexin V ⁺ cells was quantified (n=4) . Shown are the representative results from three independent experiments. *P＜0.05; **P＜0.01.

Figure6: Memory B cells from STAT3 LOF patients have disrupted early BCR activation.

(A, D-F) Sorted B cells from HC and STAT3 LOF patients’ peripheral blood (P1-P4) were stimulated with mAg for varying lengths of time, fixed, permeabilized, and stained for CD27 (to mark memory B cells) and actin usingphalloidin. Images were taken using TIRFm and analyzed for B cell contact area using IRM and the MFI of BCR and actin in the contact zone. (B and G) HC and STAT3 LOF patient B cells stimulated with mAg and stained for CD27 and pCD19 were imaged using TIRFm and the MFI of pCD19 in the contact zone was measured. (C and H) HC and STAT3 LOF patient B cells stimulated with mAg and stained for CD27 and pY were imaged with TIRFm and the MFI of pY in the contact zone was measured. Shown are representative images and the average mean values (±SD) in which more than 50 cells were analyzed using NIS-Elements AR 3.2 software from 3 independent experiments. Scale bars, 2.5 mm. *P＜0.05; **P＜0.01. Mann-Whitney U test (Fig 2D-H) . (I and J) Flow cytometry data of B cell subtypes from HC and STAT3 LOF (P1-P6) and GOF (P7) patients’ PBMC (n=6) and the percentages quantified for

B, memory B, transitional B, and plasmablast cells. (K and L) Sorted B cells from HC, STAT3 LOF (P1-P4) and GOF (P7) patients’ PBMCs were stimulated with sAg for varying lengths of time and analyzed for percentages of

and memory B cells (n=4) using flow cytometry. Shown are the representative results from three independent experiments. *P＜0.05; **P＜0.01.

Figure7: STAT3 deficiency causes the hyper IgE via enhancing the miRNA146A-14-3-3σaxis.

(A) Analysis of miRNA146A and 14-3-3σ mRNA in splenic B cells from WT and STAT3 KO mice (n=6) . (B) Analysis of miRNA146A and 14-3-3σ mRNA in B cells from HC and LOF patients’ PBMC (n=6) (P1-P7) . (C) Expression of 14-3-3σ in B cells from WT and STAT3 KO mouse spleens (left panel) as wells as HC and LOF PBMC (right panel) (P4-P6) using Western blot. GAPDH was used as a loading control. (D) PCR analysis of STAT3 binding to the 14-3-3σ promoter in B cells from WT mice. IgG was used as negative control. (E) Splenic B cells from WT and STAT3 KO mice were treated with or without 10μM of R18 for 1 hour, stimulated with sAg, then lysed and probed for pAkt and pFoxo1 using Western blot. β-actin was used as a loading control. (F) WT and STAT3 KO mice were consecutively injected with or without R18 (0.2 μg per kg body weight) by IP for 14 days, then splenic B cells were isolated and stimulated with sAg, lysed and probed for pAkt and pFoxo1 using Western blot. β-actin was used as a loading control. (G-J) WT and STAT3 KO mice (n=3) were consecutively injected with or without R18 (0.2 μg per kg body weight) by IP for 14 days, and the percentage of FO and GC B cells analyzed by flow cytometry. (K and L) WT and STAT3 KO mice treated with or without R18 and non-stimulated or stimulated were measured by flow cytometry for the percentages of IgE ⁺ B cells and IgE switched B cells. (M) The titer of IgE from serum of WT and STAT3 KO mice treated with or without R18 were measured by ELISA (n=5) . Shown are the representative results from three independent experiments. *P <0. 05 and **P <0. 01.

Figure 8

(A) Analysis of STAT3 mRNA in splenic B cells from WT and STAT3 KO mice (n=4) . (B and C) Flow cytometry data of Annexin V expression in splenic B220, MZ, FO, T1 and T2 cells from WT and STAT3 KO mice and the quantified percentages of the cell populations (n=8) . (D and E) Flow cytometry data of Ki67 expression in splenic B220, MZ, FO, T1 and T2 cells from WT and STAT3 KO mice and the quantified percentages of the cell populations (n=8) . Shown are the representative results from three independent experiments. (F and G) WT and STAT3 KO mice were consecutively injected with or without R18 (0.2 μg per kg body weight) by IP for 14 days. Data shows flow cytometry analysis of MZ cells and their percentages (n=3) . Shown are the representative results from three independent experiments. *P <0 . 05 and **P <0. 01.

Examples

The present application will be explained in detail as follows.

In this study, we found that the proximal positive BCR signaling was reduced, but the negative regulator, SHIP, was enhanced in STAT3 KO B cells. The activation of WASP and resultant actin reorganization was reduced and the early activation of B cells, including BCR clustering, B cell spreading, and signalosome recruitment, was also impaired in STAT3 KO B cells. By using peripheral blood mononuclear cells (PBMC) from STAT3 LOF patients, we found that the early activation of memory B cells was also decreased, as well as the actin accumulation. Intriguingly, we found that memory B cells and plasmablasts were reduced, but

B cells were increased both in STAT3 LOF and GOF cells. Furthermore, the

B cells from STAT3 LOF were prone to differentiate into memory B cells. Mechanistically, the STAT3 KO B cells have more, and were prone to form IgE ⁺ B cells in vitro. Finally, we found that the expression of miRNA146A and 14-3-3σ was enhanced both in STAT3 KO B cells and STAT3 LOF patients.

Transcriptional factors play a crucial role in maintaining a properly functioning immune system; therefore, mutations in transcriptional factors may lead to immune dysregulation. STAT3 or Dock8 loss-of-function mutations cause immunodeficiency and overproduction of IgE, known as hyper-IgE syndrome (HIES) , of which the underlying mechanism for this disease is largely unknown. In this study, we used samples from STAT3 loss-of-function (LOF) and gain-of-function (GOF) patients along with samples from our generated STAT3 B cell specific deletion mice (STAT3 KO) to investigate the mechanism of HIES. Interestingly, we found that the peripheral B cell homeostasis in STAT3 KO mice mimicked the phenotype of STAT3 LOF patients, having decreased follicular (FO) and germinal center (GC) B cells, but increased marginal zone (MZ) , resting and activated IgE+ B cells. Furthermore, STAT3 KO B cells had reduced B cell receptor (BCR) signaling upon antigenic stimulation due to reduced BCR clustering and decreased accumulation of WASP and F-actin. Excitingly, a central hub protein, 14-3-3σ, which is essential for the increase in IgE production, was enhanced in B cells of STAT3 KO mice and STAT3 LOF patients. The increase of 14-3-3σwas associated with increased expression of the upstream mediator, miRNA146A. The inhibition of 14-3-3σ with R18 peptide in STAT3 KO mice rescued the BCR signaling, FO, GC, and IgE+ B cell differentiation to the degree of wild-type (WT) mice. Altogether, our study has established a novel regulatory pathway of STAT3-miRNA146A-14-3-3σ to regulate BCR signaling, peripheral B cell differentiation and IgE production, which can provide targets for HIES treatment.

Materials and Methods

Mice

STAT3 conditional knockout mice on a C57/BL6 background were obtained by crossing Mb1 ^Cre mice (Jackson Laboratory) with stat3 ^flox/flox mice ³⁶ (referred here as STAT3 KO mice) , stat3 ^flox/flox mice on the same genetic background were used with wild-type controls (WT) . All mice were kept in individual ventilated cages according to protocols of the Animal Center in the Children’s Hospital affiliated with Chongqing Medical University. Mice were analyzed at 8-12 weeks of age unless otherwise noted. Bone marrow, splenic single-cell suspensions, and B cells were isolated as described previously ²⁶. For treatment with R18 in vivo, WT and STAT3 KO mice were consecutively injected with R18 (0.2 μg per kg body weight) by intraperitoneal (IP) for 14 days. All animal experiments were performed according to protocols approved by the Institutional Animal Care and Usage Committee of the Children’s Hospital of Chongqing Medical University.

Patients

From 2017 to 2018, a total of 7 Chinese patients, including 6 patients with loss of STAT3 function (LOF) and 1 patient with gain of STAT3 function (GOF) were enrolled in this study (Table 1) .

Table 1. Patient information, including gender, age, and type of STAT3 mutation.

The diagnosis of STAT3 LOF and GOF patients were made based on clinical signs, symptoms, and STAT3 mutations. Healthy control subjects consisted of 7 age-matched subjects. Human peripheral blood mononuclear cells (PBMCs) were collected and B cells were isolated from human PBMCs using the EasySep ^TM Human B Cell Isolation Kit (17954; Stemcell) . Signed consent from all of the children’s parents was obtained under the approval of the Ethics Committee of the Children’s Hospital of Chongqing Medical University.

R18 Peptide Synthesis

The R18 amino acid sequence NH ₂-YGRKKKRQRRRPHCVPRDLSWLDLEANMCLP-COOH was synthesized by GL Biochem.

Cell Culture

WT and STAT3 KO mice splenic B cells (1x10 ⁵) were obtained and stimulated with 10 μg/ml of anti-mouse CD40 (BE0016-2, BioXcell) and 8 ng/ml of IL-4 (R&D Systems) in 5 ml RPMI 1640 containing 10%FCS (Hyclone) for 5 days, the cells were then analyzed for IgE expression by flow cytometry. For detecting B cell proliferation, mouse splenic B cells (1x10 ⁵) were isolated and labeled with Cell Trace Violet, then cultured with 5 mg/mL of LPS (Sigma-Aldrich) or 10 μg/ml Class B CpG oligonucleotide (ODN1826, Invivogen) in RPMI 1640 containing 10%FCS (Hyclone) , 10 mmol/L β-mercaptoethanol, and 1%streptomycin-penicillin at a volume of 200 μl/well in 96-well round bottom plates for 5 days, then analyzed by cytometry.

Flow Cytometry and Antibodies

For flow cytometry analysis, single-cell suspensions from bone marrow and spleen were isolated and incubated with Fcγ receptor (FcγR) blocking antibody (anti-mouse CD16/CD32; BD Bioscience) on ice, then stained with the following antibodies (Abs) in PBS with 2%FBS. For surface markers, the anti-mouse Abs used included FITC–anti-CD127 (135008; BioLegend) , APC–anti-CD43 (143208) , PE–anti–BP-1 (108307; BioLegend) , Brilliant Violet 510–anti-B220 (103247; BioLegend) , PE/Cy7–anti-CD24 (101822; BioLegend) , and Brilliant Violet 421–anti-IgM (406518; BioLegend) , FITC–anti-CD19 (101506; BioLegend) , FITC–anti-CD95 (554257; BD Biosciences) , FITC–anti-Annexin V (640906; BioLegend) , PE–anti-CD23 (101608; BioLegend) , PerCP/Cy5.5–anti-IgD (405710; BioLegend) , APC–anti-CD21 (123412; BioLegend) , Alexa Fluor 647–anti-GL7 (144606; BioLegend) , Brilliant Violet 421–anti-IgM (406518; BioLegend) , PE–anti-CD5 (100608; BioLegend) , APC–anti-CD11b (101212; BioLegend) . For intracellular staining, cells were fixed and permeabilized using a Foxp3 Staining Buffer Set (eBiosciense) according to the manufacturer’s instructions and stained with Ki67 (25-56698-82, ebioscience) and PE–anti–IgE (406907; BioLegend) . Anti-human Abs and reagents used to stain surface markers of human B cells included FITC-anti-CD19 (302206; BioLegend) , Pacific Blue-anti-CD38 (356628; BioLegend) , BV510-anti-Ig D (348220; BioLegend) , PE-anti-CD24 (311106; BioLegend) , AF647-anti-CD27 (302812; BioLegend) .

Detection of the levels of phosphorylated signaling proteins has been described previously ³⁷. Briefly, splenic B cells were isolated and stained with Brilliant Violet 510–anti-B220 (103247; BioLegend) , then incubated with soluble antigen (sAg) of biotin-conjugated F (ab) ₂ anti-mouse IgG for 30 minutes and streptavidin for 10 minutes, followed by induction of BCR signaling at 37℃ for varying lengths of times. After BCR activation, cells were immediately fixed with Phosflow Lyse/fix buffer (BD Biosciences) , and permeabilized with Phosflow Perm Buffer III (BD Biosciences) . Cells were stained with antibody to pWASP (S483/S484) (A300-205A; Bethyl) and F-actin (R37110; Thermo Fisher) , and for secondary antibodies Alexa Fluor 405–goat anti-rabbit IgG antibody (A-31556; Thermo Fisher) was used. Flow cytometry data were acquired on a FACS Canto (BD Biosciences) and analyzed using FlowJo software.

Total Internal Reflection Fluorescence Microscopy and Confocal Microscopy

Total internal reflection fluorescence microscopy (TIRFm) and confocal fluorescence microscopy images were obtained as previously described ²⁶. Briefly, B cells from human PBMC and mice spleens were incubated on membrane-tethered antigen (mAg) at 37 ℃ for different time points. After fixation and permeabilization, the cells were stained for pCD19 (ab203615; Abcam) , pBtk (5082S; Cell Signaling Technology, Danvers, Mass) , pY (05-321; Merck-Millipore) , pSHIP (3941S; Cell Signaling Technology, Danvers, Mass) , and pWASP (A300-205A; Bethyl Laboratories) . TIRFm analysis was performed using NIS-Elements AR 3.2 software. The B cell contact area was determined using interference reflection microscopy (IRM) images. For the mean fluorescence intensity (MFI) analysis, the background fluorescence was subtracted and MFI measured in the B cell contact zone. For each set of data, >20 individual cells from 2 or 3 independent experiments were analyzed.

For confocal microscopy analysis, splenic B cells from WT and STAT3 KO mice were isolated and stimulated with sAg by incubating cells with AF546–monobiotinylated-Fab’–anti-IgG and streptavidin at 4 ℃, then the cells were washed and warmed to 37 ℃ for different time points. After fixation and permeabilization, the cells were stained for pCD19 (ab203615; Abcam) , pBtk (5082S; Cell Signaling Technology) , pY (05-321; Merck-Millipore) , pSHIP (3941S; Cell Signaling Technology, Danvers, Mass) , and pWASP (A300-205A; Bethyl Laboratories) , then imaged by confocal microscopy and analyzed using NIS-Elements AR 3.2 software.

Immunoblotting

Immunoblot analysis was performed as described previously ³⁷. For detection of phosphorylated signaling proteins, splenic B cells from WT and STAT3 KO mice were stimulated with sAg by incubating cells with biotinylated anti-mouse IgG F (ab) ₂for 30 minutes on ice and then with streptavidin for 10 minutes on ice, the B cells were then washed and warmed to 37 ℃ for indicated times and lysed. Cell lysates were run on an SDS-PAGE gel and immunoblotted for pAkt (Ser473; Cell Signaling Technology) , pBtk (5082S; Cell Signaling Technology) , pCD19 (3571; Cell Signaling Technology) , pY (05321; Merck-Millipore) , pSHIP (3941S; Cell Signaling Technology) , pWASP (ab5278; abacm) , p-mTOR (5536S; Cell Signaling Technology) , pS6 (4856S; Cell Signaling Technology) , pFoxo1 (9461S; Cell Signaling Technology) , and pPI3K (17366S; Cell Signaling Technology) . For detection of total protein expression, human or mouse B cells were lysed and immunoblotted for Btk (8547S; Cell Signaling Technology) , SHIP (2728S; Cell Signaling Technology) , WASP (sc-13139; Santa Cruz Biotechnology) , Akt (9272S; Cell Signaling Technology) , S6 (2217S; Cell Signaling Technology) , Foxo1 (2880S; Cell Signaling Technology) , mTOR (7C10; Cell Signaling Technology) , PI3K (4292; Cell Signaling Technology) , CD19 (3574; Cell Signaling Technology) , Btk (8547; Cell Signaling Technology) , DOCK8 (sc-292124; Santa Cruz Biotechnology) , WIP (sc-271113; Santa Cruz Biotechnology) , Human/Mouse/Rat 14-3-3σ Antibody (AF4424; Bio-Techne) , and anti-mouse GAPDH and β-actin were used for loading controls. For treatment with R18 in vitro, splenic B cells from WT and KO mice were incubated with vehicle or 10 μM R18 for 1h before incubation with sAg.

Seahorse Assays

Splenic B cells isolated from WT and STAT3 KO mice were incubated with sAg using biotinylated anti-mouse IgG F (ab) ₂at 37℃for 1 hour, then cells were incubated in XF medium (nonbuffered DMEM containing 5 mM glucose, 2 mM L-glutamine and 1 mM sodium pyruvate) . An XF-24 Extracellular Flux Analyzer (Seahorse Bioscience) was used to measure the oxygen consumption rate (OCR) in response to 1 M oligomycin, 2 M fluorocarbonyl cyanide phenylhydrazone (FCCP) and 1 M rotenone.

ELISA

Serum was collected from WT and STAT3 KO mice and the level of IgE was measured by using an ELISA Kit (eBioscience) according to the manufacturer’s instructions.

Quantitative RT-PCR

To detect the difference of STAT3, miRNA146A, and 14-3-3δ mRNA expression in WT and STAT3 KO mice as well as healthy controls (HC) and LOF patients, B cells were isolated from mouse spleens and patient PBMC, then RNA was isolated by RNAPURE kit (RP1202; BioTeke) and reverse transcribed with a PrimeScript RT reagent Kit (RR037A; Takara) . The transcribed cDNA was used to analyze the expression of different genes with Advanced SYBR Green supermix (Bio-Rad) on a CFX96 Touch Real-Time System (Bio-Rad) . STAT3 (mus) F: 5′-TGTCAGATCACATGGGCTAAAT-3′; STAT3 (mus) R: 5′-GGTCGATGATATTGTCTAGCCA-3′; 14-3-3σ (mus) sigma F: 5′-AGAACCCAGCGTTACTCTCGA-3′; 14-3-3σ (mus) sigma R: 5′-CCACCACGTTCTTGTAAGCT-3′; 14-3-3σ (human) sigma F: 5′-GAAGTTGCAGCTGATTAAGGAC-3′; 14-3-3σ (human) sigma R: 5′-TCTGGATTAGTTGCATTGGCTA-3′. The miRNA146A expression was analyzed by using the Taqman MicroRNA Transcription and MicroRNA assay kits according to the manufacturer’s protocols.

Chromatin Immunoprecipitation (ChIP) Assay

ChIP studies were performed using

Enzymatic Chromatin IP Kit (Agarose Beads) (9002S; Cell Signaling Technology) according to the manufacturer’s protocol. B cells from WT C57BL/6 mice were treated with formaldehyde at room temperature for 10 minutes. Cell lysates were sonicated to generate sheared crosslinked chromatin of 100-1000 base pairs in length. Solubilized chromatin was immunoprecipitated with antibody against STAT3 (9139S; Cell Signaling Technology) and PCR measurements were performed on a Bio-Rad PCR instrument. Primers used were as follows: mouse 14-3-3σ promoter forward 5’-CACACCCACACTACCTCACA-3’ and reverse 5’-GTGGTAGTGCTGTCCAGGTG-3.

Statistical Analysis

Statistical significance was assessed by two-tailed unpaired Student’s t-test with Prism 7 software, (*p < 0.05; **p < 0.01; ***p < 0.001) , unless otherwise stated. Data are presented as the means +/-the standard deviation (SD) .

Results

STAT3 is essential for maintaining the homeostasis of peripheral B cells, but not bone marrow B cells.

To investigate whether STAT3 is required for the development of bone marrow B cells, we created STAT3 B cell specific knockout mice Mb1 ^Cre stat3 ^flox/flox (STAT3 KO) by crossing Mb1 ^Cre mice with stat3 ^flox/flox mice. The expression of stat3 mRNA was significantly reduced in STAT3 KO B cells, indicating efficient deletion (Figure 8A) . The subsets of bone marrow B cells of STAT3 KO mice were stained with BP-1 and CD24 antibodies to determine pre-pro, pro, and early-pre, while B220-IgM antibody was used to identify late-pre, immature, and recirculating B cells. We did not observe any abnormalities for all the subpopulations examined except for the frequency of recirculating B cells, which had a decreased trend (Figure 1A, B, C) . IL-7 signaling is essential for BM B cell development, however, the IL-7 receptor expression (CD127) had no difference between WT and STAT3 KO mice (Figure 1D) . This indicates that STAT3 is dispensable for the development of bone marrow B cells. We further determined how STAT3 affects the peripheral B cells by using IgM-IgD antibodies to stain the T1, T2, and follicular (FO) B cells; CD23 and CD21 antibodies to define the marginal zone (MZ) B cells; and GL7 and CD95 antibodies to identify the germinal center (GC) B cells. The percentage and number of T1 B cells was normal, but that of T2 B cells were decreased in STAT3 KO mice (Figure 1E, F, G) . Intriguingly, the percentage and number of FO was significantly decreased (Figure 1E, H) , but that of MZ B cells was increased in STAT3 KO mice (Figure 1I, J) . Furthermore, the ratio and number of spontaneous GC B cells was decreased in STAT3 KO mice (Figure 1K, L) . We examined the proliferation and apoptosis of resting state B cells by staining with Ki67 and Annexin V, and found the expression of Ki67 and Annexin V in FO, MZ, GC, T1 and T2 cells had no difference between WT and KO mice (Figure 8B-E) . Next, we found that the percentage and number of B1a cells was normal, but the ratio of B1b cells was significantly decreased (Figure 1M, N, O) . Finally, we examined the IgE staining in resting and activated B cells that were stimulated with IL-4 and anti-CD40 for 5 days. Interestingly, we found that the percentage of IgE was increased significantly in STAT3 KO B cells, both in the resting and activated state (Figure 1P-R) . We also detected the level of IgE in WT and STAT3 KO mouse serum and found the titer of IgE in STAT3 KO mice was also increased (Figure 1S) . These results suggest that STAT3 is critical in maintaining the homeostasis of peripheral B cells.

STAT3 positively regulates the proximal BCR signaling.

Since STAT3 is critical for maintaining the homeostasis of peripheral B cells, which is highly correlated with antigenic BCR signaling ^38-40, we investigated how STAT3 regulates BCR signaling. We usedconfocal microscopy to examine the effect of the loss of STAT3 on the spatiotemporal organization of CD19, a coreceptor involved in amplifying upstream BCR signaling. The colocalization between pCD19 and the BCR was significantly diminished in STAT3 KO B cells at 5 and 10 min upon sAg stimulation (Figure 2A, B) . Additionally, the level of pCD19 was decreased in STAT3 KO B cells, which was determined by immunoblotof lysates of splenic B cells stimulated with soluble antigen (sAg) for varying lengths of time (Figure 2C) . Altogether, this implies that STAT3 affects both the spatiotemporal organization and activation of CD19. Next, we examined the total levels of BCR signaling by tyrosine phosphorylated proteins (pY) and pBtk, an immediate downstream kinase of CD19, by confocal microscopy and immunoblot using similar conditions as above. The correlation coefficient between pY/pBtk and the BCR was significantly decreased at 10 min in STAT3 KO B cells (Figure 2D, E) . Furthermore, the levels of pY and pBtk were significantly decreased in STAT3 KO B cells (Figure 2F) . Lastly, we determined the effect of STAT3 deficiency on the colocalization between pSHIP and BCR as well as the activation of SHIP, a proximal negative regulator of BCR signaling using the same conditions as above. The colocalization between the BCR and pSHIP was significantly decreased in STAT3 KO B cells at 5 min (Figure 2G, H) , and the phosphorylation of SHIP was enhanced in STAT3 KO B cells (Figure 2I) . Altogether, these resultssuggest that the deficiency of STAT3 disrupts the balance of positive and negative BCR signaling.

The deficiency of STAT3reduces the accumulation of F-actin mediated by WASP and WIP as well as BCR clustering and positive signalosome recruitment.

A previous report demonstrated a correlation between STAT3 and actin in macrophage cell lines ⁴¹. Thus, we sought to determine whether the aberrant BCR signaling in STAT3 KO B cells leads to disruption of actin. To examine this, B cells stimulated with sAg were fixed, permeabilized, and stained for the actin regulator, pWASP, and F-actin using specific antibodies and phalloidin, respectively, then analyzed by confocal microscopy, immunoblot, and phosflow. Only the expression of pWASP, but not the total WASP was decreased both in immunoblot and phosflow for STAT3 KO B cells (Figure 3 A-C) , which correlated with the reduced actin accumulation. To further pursue the underlying mechanism of how STAT3 promotes the expression of pWASP, the expression of WIP, which stabilizes the phosphorylation of WASP and protects it from degradation, was examined by immunoblot. Not surprisingly, the expression of WIP was diminished in STAT3 KO murine B cells and PBMC from STAT3 LOF patients (Figure 3D) . This indicates that the decreased level of pWASP could be due to the reduced expression of WIP. DOCK8 is upstream of WASP ¹⁵ and DOCK8 deficient patients also have been reported to have HIES ⁴². Therefore, we examined DOCK8 expression in STAT3 KO mice and LOF patients. Interestingly, the expression of DOCK8 was reduced significantly in STAT3 KO murine B cells and PBMC from STAT3 LOF patients (Figure 3D) . Our previous research has shown that defective F-actin reorganization at the plasma membrane affects the velocity of BCRs and consequential BCR clustering and BCR signaling ⁴³. To investigate the impact of the loss of STAT3 on the early activation of B cells including BCR clustering and signalosome accumulation, we used total internal reflection fluorescence microscopy (TIRFm) to observe B cells stimulated with membrane-tethered antigens (mAg) . Splenic B cells activated with mAg for varying lengths of time were stained with phalloidin, pWASP, pCD19, pBtk, pY, and pSHIP. The B cell spreading as well as BCR clustering measured by the mean fluorescence intensity (MFI) in the contact zone was significantly reduced in STAT3 KO B cells (Figure 3E, G, H) . The recruitment of F-actin and pWASP was also significantly reduced in the contact zone of STAT3 KO B cells (Figure 3F, I, J) . Additionally, the recruitment of pCD19 (Figure 4A, D) , pBtk and pY (Figure 4B, E, F) signalosomes in the B cell contact zone was significantly decreased, but that of pSHIP was enhanced in STAT3 KO B cells (Figure 4C, G) . Taken together, these results suggest that the deficiency of STAT3 reduces the recruitment of F-actin mediated by WASP at the plasma membrane, which leads to reduced BCR clustering and BCR signaling that is probably due to a lack of driving force to sequester BCRs into clusters.

STAT3 deficiency reduces the PI3K-Akt-mTORC1 mediated metabolic signaling pathway.

PI3K is involved in metabolic signaling pathways and is the immediate downstream effector of Btk activation ⁴⁴. Therefore, we examined the mTORC1 and mTORC2 signaling mediated by PI3K. Firstly, STAT3 KO B cells stimulated with sAg were lysed and probed with antibodies specific for pPI3K, and it was found that the level of pPI3K was reduced (Figure 5A) . Next, we examined the mTORC2 signaling molecules including pAkt and pFoxo1 using similar conditions. The levels of pAkt and pFoxo1 were also reduced in STAT3 KO B cells upon stimulation (Figure 5A) . Additionally, the phosphorylation level of the mTOR-common subunit of mTORC1 and mTORC2 was also reduced in STAT3 KO B cells upon stimulation (Figure5A) . Finally, the activation of the downstream kinase of Akt-mTORC1 was determined by examining the pS6 level. We found that the pS6 level was reduced as well (Figure 5A) . Because mTORC activity is highly related with cell metabolism, we used Seahorse XF technology to analyze oxidative phosphorylation in real time. The oxygen-consumption rate (OCR) was used as a measurement of oxidative phosphorylation. Cells were first treated with oligomycin to block mitochondrial ATP production and then with carbonyl cyanide 4- (trifluoromethoxy) phenylhydrazone (FCCP) to induce maximal amounts of oxygen consumption and rotenone plus antimycin to inhibit the electron transport chain. After stimulation of the cells with sAg, both basal and maximal rates of respiration were lower in STAT3 KO B cells than in WT B cells (Figure 5B) . We also examined the proliferation and apoptosis of B cells pulsed with LPS or CpG by flow cytometry using Annexin V and cell trace violet staining, and found that the proliferation of STAT3 KO B cells was increased (Figure 5C, D) , but the expression of Annexin V had no difference (Figure 5E, F) . Therefore, STAT3 KO B cells are not defective in proliferation when stimulated and are not weak and dying of apoptosis. Altogether, these results imply that STAT3 deficiency inhibits the metabolism of B cells in response to BCR stimulation.

Memory B cells from STAT3 LOF patients have disrupted early activation.

The activation of CD19 is essential for the early activation of memory B cells ⁴⁵, and to determine the effect of STAT3 deficiency on the early activation of human memory B cells, we examined the BCR clustering and B cell spreading in HCs and STAT3 LOF patients’ B cells upon stimulation with mAg by TIRFm. The contact area of memory B cells (identified as CD27 ⁺) from STAT3 LOF patients was drastically decreased compared to that of HC memory B cells (Figure 6 A, D) . The BCR clustering in the contact zone of memory B cells from STAT3 LOF patients was also significantly decreased compared to that of HCs (Figure 6 A, E) . Next, we examined the actin reorganization in memory B cells by phalloidin staining and found that the actin accumulation in the contact zone of memory B cells from STAT3 LOF patients was significantly reduced (Figure 6A, F) . To further determine the effect of STAT3 deficiency on the signalosome recruitment in memory B cells, the levels of pY and pCD19 in the contact zone were examined. The levels of pY and pCD19 in the contact zone of memory B cells from STAT3 LOF patients were profoundly decreased compared to that of HCs (Figure 6B, C, G, H) . These results suggest that STAT3 deficiency blocks the early activation of memory B cells. We further analyzed the immune phenotype of STAT3 LOF and GOF patients by different combinations of antibody staining. Interestingly, we found that the percentage of memory B cells was drastically decreased in STAT3 LOF and GOF patients and that the percentage of

B cells was increased in STAT3 LOF and GOF patients (Figure 6I, J) . Intriguingly, the frequency of transitional B cells was enhanced significantly in STAT3 LOF patients but decreased dramatically in STAT3 GOF patients. Finally, the percentage of plasmablasts was decreased both in STAT3 LOF and GOF patients (Figure 6I, J) . Next, we investigated the effect of STAT3 deficiency on the transition of

B cells to memory B cells in vitro. PBMC from STAT3 LOF patients were stimulated with sAg for 5 min and stained for IgD and CD27. We found that the frequency of

B cells (IgD ⁺CD27 ^-) was significantly decreased, but that of memory B cells (IgD ^-CD27 ^-) was significantly increased in STAT3 LOF patients (Figure 6K, L) . These results indicate that STAT3 deficient

B cells are prone to form memory B cells, which may fit the hyper IgE model in STAT3 LOF patients.

STAT3 deficiency causes hyper IgE via enhancing the miRNA146A-14-3-3σ axis.

Previous research has demonstrated that miRNA146Apromotes the IgE switching via upregulating 14-3-3σ expression ¹⁸. To determine whether STAT3 regulates the miRNA146A-14-3-3σ axis, we examined the expression of miRNA146Aand14-3-3σ in B cells of STAT3 KO mice or STAT3 LOF patientsusing RT-PCR and immunoblot. Interestingly, the mRNA levels of miRNA146a and 14-3-3σ were enhanced in STAT3 KO B cells (Figure 7A) , and expression of 14-3-3σ protein was also significantly increased in the B cells of STAT3 KO mice (Figure 7C) . The phenotype found in STAT3 KO mice was recapitulated in STAT3 LOF patients (Figure 7B, C) . Mechanistically, we found by using ChIP that STAT3 regulated 14-3-3σ expression by binding to the promoter of 14-3-3σ (Figure 7D) . To further confirm that STAT3 regulates BCR signaling, IgE production, and B cell differentiation via 14-3-3σ as the central hub, STAT3 KO B cells or mice were treated with 14-3-3σ inhibitor-peptide R18 in vitro or in vivo, respectively. We found that the levels of BCR signaling molecules, such as pAkt and pFoxo1 were rescued to the degree of WT B cells in STAT3 KO B cells (Figure 7E, F) . Furthermore, we analyzed the percentage of peripheral B cell subsets, IgE ⁺ B cells, and IgE serum in STAT3 KO mice treated with 14-3-3σ inhibitor and found the percentage of FO (Figure 7G, H) , GC (Figure 7I, J) , and IgE ⁺ B cells (Figure 7K, L) in the resting and activated state were all rescued to the degree of WT mice, except for MZ B cells (Figure 8F, G) . Additionally, the titer of IgE in serum (Figure 7M) in STAT3 KO mice injected with 14-3-3σ inhibitor was also rescued. These results indicate that STAT3-miRNA146A-14-3-3σ axis is important for the BCR signaling, IgE production, and FO and GC B cell differentiation.

Discussion

STAT3 deficiency causes HIES, but the underlying molecular mechanism is unclear. By using a STAT3 deleted mouse model and patient samples for studying the early stages of B cell development, we found that the differentiation of FO, MZ, and GC B cells was disrupted. Excitingly, the generation of IgE ⁺ B cells and isotype switching of IgE in vitro was enhanced in STAT3 KO mice, thus proving to be an optimal model for investigating the molecular mechanism of hyper-IgE caused by STAT3 deficiency. Furthermore, we found that STAT3 breaks the balance of positive and negative BCR signaling and reduces the BCR signaling. Mechanistically, we found that the BCR clustering, B cell spreading, and BCR signalosome recruitment was reduced in

B cells, which may have been due to a lack of driving force from actin. Additionally, the early activation of memory B cells including B cell spreading, BCR clustering, and BCR signalosome recruitment was also reduced in STAT3 deficient cells. Mechanistically, we found that the pivotal factor deciding the IgE isotype switching was enhanced14-3-3σ expression in STAT3 KO B cells, as well as its upstream mediator miRNA146A. Interestingly, we found that the inhibition of 14-3-3σ in vitro or in vivo can rescue the BCR signaling and IgE production in STAT3 KO B cells or STAT3 KO mice to normal levels. This is the first time to be reported that there is enhanced 14-3-3σ expression in STAT3 KO B cells.

The role of 14-3-3σ has been studied in B cells before. 14-3-3σ deficient mice have reduced peripheral B cells such as FO, MZ, and recirculating B cells and T cell independent antigen responses ⁴⁶. In STAT3 KO mice, the MZ B cells were reduced although the 14-3-3σexpression was enhanced, which suggests that optimal 14-3-3σ expression is critical for the homeostasis of peripheral B cells. 14-3-3σ deficient mice have reduced pAkt and pErk1/2 signaling upon stimulation with anti-IgM ⁴⁶. In our STAT3 KO mice, the proximal and distal BCR signaling was also reduced upon antigenic stimulation. Again, this indicates the optimal expression of 14-3-3σ is important for normal BCR signaling.

The relationship between BCR signaling and IgE production still remains elusive. DOCK8 patients have HIES and we found that the level of BCR signaling is reduced in DOCK8 deficient B cells ¹⁵. Similarly, in STAT3 patients, we also found a reduction of DOCK8 expression and decreased levels of BCR signaling. These results imply that the decreased BCR signaling may induce the IgE isotype switching. Previous research has shown that BCR cross-linking blocks the class switching of IgG1 and IgE by adding LPS with IL-4 ⁴⁷.

In our study, the inhibition of BCR signaling in STAT3 KO B cells with 14-3-3σinhibitor, R18, decreases the IgE production. Therefore, we established that the low BCR signaling offers an advantage for the IgE isotype switching. We have also shown that STAT3 expression is essential for the early activation of memory B cells. Intriguingly, the LOF and GOF of STAT3 patients both have reduced memory B cells and increased

B cells, which indicates that the optimal expression of STAT3 is critical to maintain the memory response of B cells.

One remaining issue is the detailed molecular mechanism of how STAT3 regulates BCR signaling. The transcriptional levels of CD19, BTK, and SHIP were not altered at all in STAT3 KO B cells, which indicates that STAT3 does not regulate the transcription of the proximal BCR signaling genes. It would be crucial to use ChIP-assay and STAT3 antibody as bait to find the regulatory regions of the target genes. STAT3 has been shown to interact with several proteins such as NF-kB, RelA, HIF1a, Jun, STAT1, mTOR, and ICOS ^26, 48-53.

One paradox is that there are reduced memory B cells in STAT3 patients, but somehow STAT3 KO

B cells are prone to forming memory B cells, which could be due to a compensation mechanism. Previous research has shown that memory B cells might need less STAT3 activity to respond to cytokines to go through plasmablast differentiation ³⁵. Our results have demonstrated that STAT3 KO

B cells are prone to becoming memory B cells and IgE switching plasmablasts; this could be due to higher expression of miRNA146A and 14-3-3σ protein. We will need to further check the expression of activation-induced deaminase (AID) in STAT3 KO B cells. But it is still puzzling why STAT3 patients have reduced memory B cells. A recent study has shown that mTORC1 deficiency leads to reduced Ag-specific memory B cells as well as plasma cells and GC B cells ⁵⁴. Our results have shown that both mTORC1 and mTORC2 activity is reduced in STAT3 KO B cells and the STAT3-mTORC axis regulates the formation of memory B cells. Additionally, STAT3 KO naive B cells have low metabolic activity, which may be the reason to switch into memory B cells from

B cells.

Overall, our study has revealed the underlying mechanism for the hyper IgE syndrome in STAT3 patients by using an optimal mouse model and patient samples. The enhanced expression of 14-3-3σ in STAT3 KO mice caused increased generation of IgE ⁺ B cells and lower BCR signaling.

References

1. Yuan ZL, Guan YJ, Wang L, Wei W, Kane AB, Chin YE. Central role of the threonine residue within the p+1 loop of receptor tyrosine kinase in STAT3 constitutive phosphorylation in metastatic cancer cells. Mol Cell Biol 2004; 24: 9390-400.

2. Takeda K, Noguchi K, Shi W, Tanaka T, Matsumoto M, Yoshida N, et al. Targeted disruption of the mouse Stat3 gene leads to early embryonic lethality. Proc Natl Acad Sci U S A 1997; 94: 3801-4.

3. Yang XO, Panopoulos AD, Nurieva R, Chang SH, Wang D, Watowich SS, et al. STAT3 regulates cytokine-mediated generation of inflammatory helper T cells. J Biol Chem 2007; 282: 9358-63.

4. Ma CS, Chew GY, Simpson N, Priyadarshi A, Wong M, Grimbacher B, et al. Deficiency of Th17 cells in hyper IgE syndrome due to mutations in STAT3. J Exp Med 2008; 205: 1551-7.

5. Renner ED, Rylaarsdam S, Anover-Sombke S, Rack AL, Reichenbach J, Carey JC, et al. Novel signal transducer and activator of transcription 3 (STAT3) mutations, reduced T(H) 17 cell numbers, and variably defective STAT3 phosphorylation in hyper-IgE syndrome. J Allergy Clin Immunol 2008; 122: 181-7.

6. Al Khatib S, Keles S, Garcia-Lloret M, Karakoc-Aydiner E, Reisli I, Artac H, et al. Defects along the T (H) 17 differentiation pathway underlie genetically distinct forms of the hyper IgE syndrome. J Allergy Clin Immunol 2009; 124: 342-8, 8 e1-5.

7. McIlwain DR, Grusdat M, Pozdeev VI, Xu HC, Shinde P, Reardon C, et al. T-cell STAT3 is required for the maintenance of humoral immunity to LCMV. Eur J Immunol 2015; 45: 418-27.

8. Levy DE, Loomis CA. STAT3 signaling and the hyper-IgE syndrome. N Engl J Med 2007; 357: 1655-8.

9. Minegishi Y, Saito M, Tsuchiya S, Tsuge I, Takada H, Hara T, et al. Dominant-negative mutations in the DNA-binding domain of STAT3 cause hyper-IgE syndrome. Nature 2007; 448: 1058-62.

10. Milner JD, Vogel TP, Forbes L, Ma CA, Stray-Pedersen A, Niemela JE, et al. Early-onset lymphoproliferation and autoimmunity caused by germline STAT3 gain-of-function mutations. Blood 2015; 125: 591-9.

11. Zhang Q, Davis JC, Lamborn IT, Freeman AF, Jing H, Favreau AJ, et al. Combined immunodeficiency associated with DOCK8 mutations. N Engl J Med 2009; 361: 2046-55.

12. Yang L, Fliegauf M, Grimbacher B. Hyper-IgE syndromes: reviewing PGM3 deficiency. Curr Opin Pediatr 2014; 26: 697-703.

13. Minegishi Y, Saito M, Morio T, Watanabe K, Agematsu K, Tsuchiya S, et al. Human tyrosine kinase 2 deficiency reveals its requisite roles in multiple cytokine signals involved in innate and acquired immunity. Immunity 2006; 25: 745-55.

14. Bitoun E, Chavanas S, Irvine AD, Lonie L, Bodemer C, Paradisi M, et al. Netherton syndrome: disease expression and spectrum of SPINK5 mutations in 21 families. J Invest Dermatol 2002; 118: 352-61.

15. Sun X, Wang J, Qin T, Zhang Y, Huang L, Niu L, et al. Dock8 regulates BCR signaling and activation of memory B cells via WASP and CD19. Blood Adv 2018; 2: 401-13.

16. Jabara HH, McDonald DR, Janssen E, Massaad MJ, Ramesh N, Borzutzky A, et al. DOCK8 functions as an adaptor that links TLR-MyD88 signaling to B cell activation. Nat Immunol 2012; 13: 612-20.

17. Lam T, Thomas LM, White CA, Li G, Pone EJ, Xu Z, et al. Scaffold functions of 14-3-3 adaptors in B cell immunoglobulin class switch DNA recombination. PLoS One 2013; 8: e80414.

18. Li F, Huang Y, Huang YY, Kuang YS, Wei YJ, Xiang L, et al. MicroRNA-146a promotes IgE class switch in B cells via upregulating 14-3-3sigma expression. Mol Immunol 2017; 92: 180-9.

19. Xu Z, Fulop Z, Wu G, Pone EJ, Zhang J, Mai T, et al. 14-3-3 adaptor proteins recruit AID to 5'-AGCT-3'-rich switch regions for class switch recombination. Nat Struct Mol Biol 2010; 17: 1124-35.

20. Saki N, Abroun S, Soleimani M, Mortazavi Y, Kaviani S, Arefian E. The roles of miR-146a in the differentiation of Jurkat T-lymphoblasts. Hematology 2014; 19: 141-7.

21. Sun X, Zhang J, Hou Z, Han Q, Zhang C, Tian Z. miR-146a is directly regulated by STAT3 in human hepatocellular carcinoma cells and involved in anti-tumor immune suppression. Cell Cycle 2015; 14: 243-52.

22. Ren JP, Ying RS, Cheng YQ, Wang L, El Gazzar M, Li GY, et al. HCV-induced miR146a controls SOCS1/STAT3 and cytokine expression in monocytes to promote regulatory T-cell development. J Viral Hepat 2016; 23: 755-66.

23. Liu W, Meckel T, Tolar P, Sohn HW, Pierce SK. Intrinsic properties of immunoglobulin IgG1 isotype-switched B cell receptors promote microclustering and the initiation of signaling. Immunity 2010; 32: 778-89.

24. Tolar P, Hanna J, Krueger PD, Pierce SK. The constant region of the membrane immunoglobulin mediates B cell-receptor clustering and signaling in response to membrane antigens. Immunity 2009; 30: 44-55.

25. Tolar P, Sohn HW, Pierce SK. The initiation of antigen-induced B cell antigen receptor signaling viewed in living cells by fluorescence resonance energy transfer. Nat Immunol 2005; 6: 1168-76.

26. Huang L, Zhang Y, Xu C, Gu X, Niu L, Wang J, et al. Rictor positively regulates B cell receptor signaling by modulating actin reorganization via ezrin. PLoS Biol 2017; 15: e2001750.

27. Seeley-Fallen MK, Liu LJ, Shapiro MR, Onabajo OO, Palaniyandi S, Zhu X, et al. Actin-binding protein 1 links B-cell antigen receptors to negative signaling pathways. Proc Natl Acad Sci U S A 2014; 111: 9881-6.

28. Liu C, Miller H, Hui KL, Grooman B, Bolland S, Upadhyaya A, et al. A balance of Bruton's tyrosine kinase and SHIP activation regulates B cell receptor cluster formation by controlling actin remodeling. J Immunol 2011; 187: 230-9.

29. Sharma S, Orlowski G, Song W. Btk regulates B cell receptor-mediated antigen processing and presentation by controlling actin cytoskeleton dynamics in B cells. J Immunol 2009; 182: 329-39.

30. Chou WC, Levy DE, Lee CK. STAT3 positively regulates an early step in B-cell development. Blood 2006; 108: 3005-11 %U http: //www. bloodjournal. org/cgi/doi/10.1182/blood-2006-05-024430.

31. Fornek JL, Tygrett LT, Waldschmidt TJ, Poli V, Rickert RC, Kansas GS. Critical role for Stat3 in T-dependent terminal differentiation of IgG B cells. Blood 2006; 107: 1085-91.

32. Ding C, Chen X, Dascani P, Hu X, Bolli R, Zhang HG, et al. STAT3 Signaling in B Cells Is Critical for Germinal Center Maintenance and Contributes to the Pathogenesis of Murine Models of Lupus. J Immunol 2016; 196: 4477-86.

33. Speckmann C, Enders A, Woellner C, Thiel D, Rensing-Ehl A, Schlesier M, et al. Reduced memory B cells in patients with hyper IgE syndrome. Clin Immunol 2008; 129: 448-54.

34. Meyer-Bahlburg A, Renner ED, Rylaarsdam S, Reichenbach J, Schimke LF, Marks A, et al.Heterozygous signal transducer and activator of transcription 3 mutations in hyper-IgE syndrome result in altered B-cell maturation. J Allergy Clin Immunol 2012; 129: 559-62, 62 e1-2.

35. Deenick EK, Avery DT, Chan A, Berglund LJ, Ives ML, Moens L, et al. Naive and memory human B cells have distinct requirements for STAT3 activation to differentiate into antibody-secreting plasma cells. J Exp Med 2013; 210: 2739-53.

36. Takeda K, Kaisho T, Yoshida N, Takeda J, Kishimoto T, Akira S. Stat3 activation is responsible for IL-6-dependent T cell proliferation through preventing apoptosis: generation and characterization of T cell-specific Stat3-deficient mice. J Immunol 1998; 161: 4652-60.

37. Jing Y, Kang D, Liu L, Huang H, Chen A, Yang L, et al. Dedicator of cytokinesis protein 2 couples with lymphoid enhancer-binding factor 1 to regulate expression of CD21 and B-cell differentiation. J Allergy Clin Immunol 2019.

38. Pillai S, Cariappa A. The follicular versus marginal zone B lymphocyte cell fate decision. Nat Rev Immunol 2009; 9: 767-77.

39. Cariappa A, Tang M, Parng C, Nebelitskiy E, Carroll M, Georgopoulos K, et al. The follicular versus marginal zone B lymphocyte cell fate decision is regulated by Aiolos, Btk, and CD21. Immunity 2001; 14: 603-15.

40. Thomas MD, Srivastava B, Allman D. Regulation of peripheral B cell maturation. Cell Immunol 2006; 239: 92-102.

41. Carruthers NJ, Stemmer PM, Chen B, Valeriote F, Gao X, Guatam SC, et al. Phosphoproteome and transcription factor activity profiling identify actions of the anti-inflammatory agent UTL-5g in LPS stimulated RAW 264.7 cells including disrupting actin remodeling and STAT-3 activation. Eur J Pharmacol 2017; 811: 66-73.

42. Grimbacher B, Holland SM, Puck JM. Hyper-IgE syndromes. Immunol Rev 2005; 203: 244-50.

43. Song W, Liu C, Upadhyaya A. The pivotal position of the actin cytoskeleton in the initiation and regulation of B cell receptor activation. Biochim Biophys Acta 2014; 1838: 569-78.

44. Kikuchi H, Kuribayashi F, Takami Y, Imajoh-Ohmi S, Nakayama T. GCN5 regulates the activation of PI3K/Akt survival pathway in B cells exposed to oxidative stress via controlling gene expressions of Syk and Btk. Biochem Biophys Res Commun 2011; 405: 657-61.

45. Depoil D, Fleire S, Treanor BL, Weber M, Harwood NE, Marchbank KL, et al. CD19 is essential for B cell activation by promoting B cell receptor-antigen microcluster formation in response to membrane-bound ligand. Nat Immunol 2008; 9: 63-72.

46. Su YW, Hao Z, Hirao A, Yamamoto K, Lin WJ, Young A, et al. 14-3-3sigma regulates B-cell homeostasis through stabilization of FOXO1. Proc Natl Acad Sci U S A 2011; 108: 1555-60.

47. Jabara HH, Chaudhuri J, Dutt S, Dedeoglu F, Weng Y, Murphy MM, et al. B-cell receptor cross-linking delays activation-induced cytidine deaminase induction and inhibits class-switch recombination to IgE. J Allergy Clin Immunol 2008; 121: 191-6 e2.

48. Ysebrant de Lendonck L, Eddahri F, Delmarcelle Y, Nguyen M, Leo O, Goriely S, et al. STAT3 signaling induces the differentiation of human ICOS (+) CD4 T cells helping B lymphocytes. PLoS One 2013; 8: e71029.

49. Jung JE, Kim HS, Lee CS, Shin YJ, Kim YN, Kang GH, et al. STAT3 inhibits the degradation of HIF-1alpha by pVHL-mediated ubiquitination. Exp Mol Med 2008; 40:479-85.

50. Zhang X, Wrzeszczynska MH, Horvath CM, Darnell JE, Jr. Interacting regions in Stat3 and c-Jun that participate in cooperative transcriptional activation. Mol Cell Biol 1999; 19: 7138-46.

51. Yu Z, Zhang W, Kone BC. Signal transducers and activators of transcription 3 (STAT3) inhibits transcription of the inducible nitric oxide synthase gene by interacting with nuclear factor kappaB. Biochem J 2002; 367: 97-105.

52. Xia L, Wang L, Chung AS, Ivanov SS, Ling MY, Dragoi AM, et al. Identification of both positive and negative domains within the epidermal growth factor receptor COOH-terminal region for signal transducer and activator of transcription (STAT) activation. J Biol Chem 2002; 277: 30716-23.

53. Yokogami K, Wakisaka S, Avruch J, Reeves SA. Serine phosphorylation and maximal activation of STAT3 during CNTF signaling is mediated by the rapamycin target mTOR. Curr Biol 2000; 10: 47-50.

54. Raybuck AL, Cho SH, Li J, Rogers MC, Lee K, Williams CL, et al. B Cell-Intrinsic mTORC1 Promotes Germinal Center-Defining Transcription Factor Gene Expression, Somatic Hypermutation, and Memory B Cell Generation in Humoral Immunity. J Immunol 2018; 200: 2627-39.

Claims

A method for prevention and/or treatment of a disease associated with STAT3 in a patient in need of such prevention and/or treatment, comprising regulation of 14-3-3σ in the patient.
The method according to Claim 1, wherein the STAT3 is STAT3 with loss of function or STAT3 with gain of function.
The method according to Claim 1 or 2, wherein the disease associated with STAT3 is hyper-IgE syndrome.
The method according to any one of Claims 1-3,

wherein the regulation of 14-3-3σ is down-regulation of 14-3-3σ or up-regulation of 14-3-3σ,

optionally wherein the down-regulation of 14-3-3σ is conducted with an inhibitor of 14-3-3σ,

optionally wherein the inhibitor of 14-3-3σ is a peptide, and

optionally wherein the peptide comprises the amino acid sequence NH ₂-YGRKKKRQRRRPHCVPRDLSWLDLEANMCLP-COOH.
A regulator of 14-3-3σ, a pharmaceutical composition comprising the regulator of 14-3-3σ or a kit comprising the regulator of 14-3-3σ, for use in prevention and/or treatment of a disease associated with STAT3 in a patient in need of such prevention and/or treatment.
The regulator, pharmaceutical composition or kit according to Claim 5,

wherein the regulator of 14-3-3σ is a down-regulator of 14-3-3σ or an up-regulator of 14-3-3σ,

optionally wherein the down-regulator of 14-3-3σ is an inhibitor of 14-3-3σ,

optionally wherein the inhibitor of 14-3-3σ is a peptide, and

optionally wherein the peptide comprises the amino acid sequence NH ₂-YGRKKKRQRRRPHCVPRDLSWLDLEANMCLP-COOH.
The regulator, pharmaceutical composition or kit according to Claim 5 or 6, wherein the STAT3 is STAT3 with loss of function or STAT3 with gain of function.
The regulator, pharmaceutical composition or kit according to any one of Claims 5-7, wherein the disease associated with STAT3 is hyper-IgE syndrome.
Use of a regulator of 14-3-3σ in prevention and/or treatment of a disease associated with STAT3 in a patient in need of such prevention and/or treatment.
Use of a regulator of 14-3-3σ in manufacture of a medicament for prevention and/or treatment of a disease associated with STAT3 in a patient in need of such prevention and/or treatment.
The use according to Claim 9 or 10,

wherein the regulator of 14-3-3σ is a down-regulator of 14-3-3σ or an up-regulator of 14-3-3σ,

optionally wherein the down-regulator of 14-3-3σ is an inhibitor of 14-3-3σ,

optionally wherein the inhibitor of 14-3-3σ is a peptide, and

optionally wherein the peptide comprises the amino acid sequence NH ₂-YGRKKKRQRRRPHCVPRDLSWLDLEANMCLP-COOH.
The use according to any one of Claims 9-11, wherein the STAT3 is STAT3 with loss of function or STAT3 with gain of function.
The use according to any one of Claims 9-12, wherein the disease associated with STAT3 is hyper-IgE syndrome.