Modulation of the humoral immune response by constitutively active STAT6 expression in murine B cells

The transcription factor STAT6 regulates gene expression in response to IL‐4 and IL‐13. To further investigate how activated STAT6 modulates B cells development and function in vivo, we characterized mice that express a constitutively active version specifically in B cells. CD19Cre_STAT6vt mice show spontaneous phosphorylation and nuclear translocation of STAT6 in B cells. About 80 genes were more than twofold up‐ or downregulated in splenic B cells from CD19Cre_STAT6vt as compared to control mice. B cell development, tissue localization, and populations of follicular and marginal zone B cells, B1 B cells, GC B cells, and plasma cells (PCs) appeared to be normal. However, the number of IgE+ and IgG1+ GC B cells and PCs as well as serum IgE and IgG1 levels were increased in CD19Cre_STAT6vt mice. Infection with Lymphocytic choriomeningitis virus associated with high levels of TNF and IFN‐γ did not prevent the development of a significantly increased IgE and IgG1 response against the virus in CD19Cre_STAT6vt mice. These results suggest that prolonged STAT6 activation during chronic allergic inflammation may result in IgE responses during subsequent viral or bacterial infection that could further stimulate mast cell activation even in the absence of the initial allergic response.


Introduction
The transcription factor signal transducer and activator of transcription (STAT6) mediates signaling downstream of the receptors for IL-4 and IL-13 [1]. After receptor activation, monomeric STAT6 gets recruited by its SH2 domain and binds to phosphotyrosine residues in the cytoplasmic domain of the IL-4Rα chain. STAT6 gets phosphorylated at Tyr 641 by Janus kinases Jak1 or Jak3, forms homodimers and translocates to the nucleus where it binds to the spaced palindromic sequence TTCN [3][4] GAA (also Correspondence: Prof. David Voehringer e-mail: David.voehringer@uk-erlangen.de named GAS-4) and thereby regulates gene expression [2]. In B cells, STAT6 is required for class switch recombination to IgG1 and IgE [3][4][5][6]. We recently performed a comparative analysis of the STAT6-regulated transcriptome and proteome in primary mouse B cells and revealed that upon stimulation with IL-4 more than 400 genes were up-or downregulated more than threefold in a STAT6dependent manner [7]. Using mixed BM chimeras, we could further show that STAT6 expression in B cells was required for GC formation during type 2 immune responses against helminths, model allergens, or sheep RBCs [8].
A constitutively active version of STAT6 (STAT6vt) was generated by alanine scanning mutagenesis of the SH2 domain [9]. The exchange of Val 547 and Thr 548 to Ala resulted in spontaneous Jak1/Jak3-independent phosphorylation of the critical Tyr 641 residue required for dimerization [10]. To further investigate the in vivo effects of STAT6vt in lymphocytes, transgenic mice were generated that express STAT6vt under control of the human CD2 promoter and locus control region that leads to expression in T and B cells. These CD2:STAT6vt mice showed reduced numbers of peripheral T cells, a Th2 bias, more B cells and higher serum levels of IgG1 and IgE [11]. Since in this model STAT6vt is expressed in all lymphocytes, it was impossible to dissect T and B cell-intrinsic effects of STAT6vt. Furthermore, it has not been investigated whether pSTAT6 in B cells impairs the usual Th1driven and IgG2a/c-biased humoral immune response to a viral infection.
To address these points, we first generated mice that express STAT6vt under control of a loxP-flanked STOP cassette from the Rosa26 locus [12] and crossed them to CD19Cre mice [13] so that expression is restricted to the B cell lineage. We show that STAT6 is constitutively phosphorylated and detectable in the nucleus of B cells from CD19Cre STAT6vt mice. RNAseq analysis revealed that 57 genes were more than twofold upregulated and 21 genes were more than twofold downregulated. B cell development, B cell numbers, and their localization in B cell follicles and in the marginal zone in the spleen appeared to be normal. All splenic B cells expressed more CD23 while CD86 expression was only increased in GC B cells. Serum IgE and IgG1 levels were increased in CD19Cre STAT6vt as compared to control mice. Infection with lymphocytic choriomeningitis virus (LCMV) revealed that constitutively active STAT6 in B cells promotes a IgG1/IgE response to the virus but does not inhibit the IgG2c response.

Expression of constitutively active STAT6 in B cells of CD19Cre STAT6vt mice
B cell-intrinsic expression of STAT6 plays an important role for class switch recombination to IgG1 and IgE but also for GC formation during type 2 immune responses. To further investigate the impact of STAT6 signaling on B cell biology, we generated CD19Cre STAT6vt mice that express a constitutively active form of STAT6 specifically in B cells. The phosphorylation status of STAT6 was determined by western blot analysis with untouched splenic B cells of WT, STAT6-deficient, and CD19Cre STAT6vt mice after purification by magnetic cell sorting and culture for up to 4 h either with LPS alone or with LPS and IL-4. The expression level of total STAT6 was comparable between B cells of WT and CD19Cre STAT6vt mice and did not change over time in response to LPS alone or LPS and IL-4 (Fig. 1A). As expected, phosphorylated STAT6 (pSTAT6) was detected in unstimulated and LPSstimulated samples of CD19Cre STAT6vt mice but not in WT and STAT6-deficient mice ( Fig. 1A; Supporting Information Fig. 1). Next, we performed histological staining to determine whether STAT6 spontaneously localizes to the nucleus of B cells in CD19Cre STAT6vt mice. In comparison to WT B cells, we observed a similar cytoplasmic but more pronounced nuclear staining in samples from CD19Cre STAT6vt mice (Fig. 1B).
These experiments clearly demonstrate that STAT6vt is spontaneously phosphorylated and translocates to the nucleus in B cells of CD19Cre STAT6vt mice.

Transcriptome analysis of splenic B cells from naïve CD19Cre STAT6vt and control mice
Based on the finding that STAT6vt accumulates in the nucleus of naïve splenic B cells in CD19Cre STAT6vt mice, we next determined the changes of the global transcriptome by RNAseq analysis. Therefore, we isolated naïve B cells by cell sorting (CD19 + CD3 neg PI neg , purity >95%) from three age-matched CD19Cre STAT6vt and three CD19Cre control mice, purified total RNA, and performed transcriptome analysis using the Illumina platform. Setting a p-value cutoff at 0.05 we revealed 973 differentially expressed genes ( Fig. 2A). Fifty-seven genes were more than twofold upregulated and 21 genes were more than twofold downregulated (Fig. 2B). Among the 36 upregulated genes with a combined expression level >1 were IgE and IgG1, the LIF receptor, Cathepsin E, PIANP (a ligand for the inhibitory receptor PILRa), PTPRV (a tyrosine phosphatase), and the signaling adaptor SLP-76 ( Fig. 2C; Supporting Information Fig.  2). Some of the 11 more than twofold downregulated genes with a combined expression level >1 included two miRNAs (Mir421 and Gm27663), KLRA1 (an inhibitory receptor), and a few uncharacterized genes ( Fig. 2B and C). Results were compared our results to published data on gene expression of in vitro IL-4-stimulated primary mouse B cells from WT and STAT6ko mice [7,14]. Gene set enrichment analysis provides evidence that a similar pattern of genes is upregulated by activated WT STAT6 and STAT6vt (Fig. 2D). Gene set enrichment analysis was also performed to screen for changes in hallmark pathways (MSigDB database) and all eight gene sets with a false discovery rate <0.05 were upregulated. Included were cell cycle related gene sets as well as Myc, MTORC, glycolysis, angiogenesis, and unfolded protein response related gene sets (Fig. 2E).

Constitutively active STAT6 does not affect B cell development and homeostasis
To further investigate whether expression of STAT6vt changes B cell development in the BM or B cell homeostasis in spleen and peritoneum, we performed flow cytometric analysis. In the BM, the frequency of pre/pro B cells (B220 lo IgM neg ), immature B cells (B220 hi IgM int ), and mature B cells (B220 lo IgM hi ) was comparable between CD19Cre STAT6vt and CD19Cre control mice (Fig. 3A). In the spleen, the total B cell frequency and the fraction Next, we determined the expression levels of the low-affinity IgE receptor CD23, the co-stimulatory molecule CD86 and MHC class II (I-A b ) as these proteins have been shown to be upregulated on B cells from CD2:STAT6vt mice that express constitutively active STAT6 in all lymphocytes under control of the human CD2 promoter [11]. Ex vivo isolated splenic B cells from naïve CD19Cre STAT6vt mice showed strongly increased expression of CD23 on all B cells while CD86 was only upregulated on B cells with a GC B cells phenotype (B220 + CD38 lo GL-7 hi ) ( Fig. 3B; Supporting Information Fig. 3). However, the expression level of MHC-II was comparable between CD19Cre STAT6vt and CD19Cre control mice (Fig. 3B). Similar results were obtained with B cells from LCMV infected mice (Supporting Information Fig. 4). Upon stimulation with LPS+IL-4 splenic B cells from CD19Cre STAT6vt mice showed more pronounced upregulation of CD23, CD86, and MHC-II on the cell surface as compared to B cells from CD19Cre control mice (Supporting Information Fig. 5).
Histological analysis of cryosections from the spleen by immunofluorescence staining revealed no obvious structural differences of B cell follicles and marginal zone B cells (Fig. 3C).
Overall, we did not observe major effects on B cell development and homeostasis by expression of STAT6vt in B cells.

GC B cells and PCs express more IgG1 and IgE in naïve and LCMV-infected CD19Cre STAT6vt mice
Since STAT6 signaling is not only important for CSR to IgG1 and IgE, but also required for GC formation during type 2 immune responses, we next analyzed the GC B cell and PC compartments and expression of IgE, IgG1, and IgG2c within these cells. The analysis was performed with naïve and LCMV-infected CD19Cre STAT6vt and CD19Cre control mice. The LCMV infection was chosen to determine whether an IgE/IgG1 response could be observed even during a strong type 1 immune response with high IFN-γ levels that usually inhibit CSR to IgE and IgG1.
Naïve CD19Cre STAT6vt mice showed no spontaneous expansion of the splenic GC B cell population (B220 + CD38 lo GL-7 hi ).  However, the frequency of IgG1 + cells within the GC B cell compartment was increased from 10 to 30% while IgG2c + and IgE + GC B cells were comparable in both strains ( Fig. 4A and E). The GC B cell population increased from below 1% in naïve mice to 4-5% on day 14 after LCMV infection in both CD19Cre STAT6vt and CD19Cre control mice (Fig. 4B). Within the GC B cell compartment of LCMV-infected mice the IgG1 + cells constituted 4% in CD19Cre and 8% in CD19Cre STAT6vt mice. The frequency of IgE + GC B cells was generally very low but still increased in CD19Cre STAT6vt mice, while the frequency of IgG2c + GC B cells was around 17% in both strains of mice ( Fig. 4B and E; Supporting Imformation Fig. S6A and B). Within the PC compartment (B220 neg CD138 + TACI + ) of naïve mice IgG1 + cells increased from 0.5% in CD19Cre mice to 2.5% in CD19Cre STAT6vt mice ( Fig. 4C and F). Likewise IgE + PCs increased from 0.2 to 0.5% while IgG2c + PCs were not significantly different ( Fig. 4C and F; Supporting Information Fig. 6C). After LCMV infection IgG1 + cells constituted 20% of all PCs in CD19Cre mice and about 32% in CD19Cre STAT6vt mice (Fig. 4C and F). The frequency of IgE + PCs increased from 0.1% in CD19Cre mice to 0.8% in CD19Cre STAT6vt mice while IgG2c + PCs were comparable with about 17% in both strains of LCMV infected mice ( Fig. 4C and F; Supporting Information Fig. 6D).

B cell-intrinsic STAT6 signaling promotes LCMV-specific IgE and IgG1 production
The significantly increased frequencies of IgE + and IgG1 + PCs after LCMV infection in CD19Cre STAT6vt mice suggested that this difference results in higher serum IgE and IgG1 levels as compared to control mice. ELISA analyses for total serum concentrations of different isotypes revealed that naïve CD19Cre STAT6vt mice produced about threefold more total IgE while IgG1 levels were only slightly increased (Fig. 5A). IgM and IgG2c levels were comparable between CD19Cre STAT6vt and control mice. Interestingly, the increased serum IgE and IgG1 levels were also detected after LCMV infection (Fig. 5A). To further determine whether the IgE/IgG1 response included LCMV-specific antibodies, we performed ELISAs specific for LCMV GP. Indeed, significantly higher serum levels of LCMV-GP-specific IgE and IgG1 antibodies were detected in CD19Cre STAT6vt as compared to control mice (Fig. 5B).
These results indicate that specific expression of constitutively active STAT6 in B cells promotes the humoral type 2 immune response even during a strongly type 1 immunity biased response to LCMV infection.

Discussion
The requirement of STAT6 for IL-4-induced CSR to IgE and IgG1 in B cells is well established [2-4, 7, 14, 15]. In addition, B cell intrinsic expression of STAT6 appears to be critical for GC formation during type 2 immune responses [8]. These findings are mainly based on analyses of STAT6-deficient B cells. However, it remained unclear whether ectopic expression of active STAT6 in B cells would be sufficient for spontaneous IgE/IgG1 CSR and GC formation. Therefore, we investigated here the phenotype of CD19Cre STAT6vt mice in which mature B cells express a constitutively active form of STAT6 from the Rosa26 locus.
Analysis of B cells isolated from naïve CD19Cre STAT6vt mice revealed the spontaneous phosphorylation and nuclear translocation of STAT6vt. However, only 47 genes with a decent expression level were significantly regulated (36 up and 11 down). This stands in contrast to several hundred STAT6-regulated genes observed in ex vivo isolated B cells cultured for 4 days with LPS and anti-CD40 in presence or absence of IL-4 [7]. One possible explanation for this difference could be linked to the effects of LPS that provides a strong proliferative signal and probably makes chromatin more accessible for STAT6. In addition, some STAT6regulated genes may require additional transcription factors for their expression and LPS-or anti-CD40-induced activation of NF-κB could play an important role here. In fact, NF-κB activity has been shown to be required for DNA binding of STAT6 [16,17]. The upregulated genes in B cells of CD19Cre STAT6vt mice included the heavy chains for IgE and IgG1 indicating enhanced CSR to these isotypes. Other upregulated genes such as Gatm, Lcp2, Lifr, and Nfil3 that had been previously identified as STAT6-regulated genes in B cells [7,18]. Nfil3 has been described to promote IgE CSR while the function of most other STAT6-regulated genes in B cells remains to be analyzed [19].
Despite these differences in gene expression and several reports that identified activating STAT6 mutations in B cell lymphomas [20][21][22], we neither found obvious alterations in the composition of the major peripheral B cell populations in the spleen or peritoneal cavity nor did we observe mice that developed lymphomas. B cell development in the BM also appeared to be normal, which could have been expected based on the fact that Cre recombinase is expressed rather late during B cell development in CD19Cre mice [13]. However, we observed upregulation of CD23 on the surface of B cells from CD19Cre STAT6vt mice. Similar findings were described in CD2:STAT6vt mice and in a B cell line transduced with a construct where STAT6 can be induced to dimerize by adding tamoxifen to the culture [11,23]. In contrast to CD2:STAT6vt mice where T and B cells express STAT6vt, we did not observe upregulation of MHC-II and slightly higher levels of CD86 were only detected in GC B cells. This indicates that additional signals that may be derived from activated T cells are required for MHC-II and CD86 upregulation in B cells.
Since B cell-intrinsic STAT6 appears to be important for GC formation during type 2 immune responses [8], we expected to see more GC B cells in CD19Cre STAT6vt as compared to control mice. However, this was not the case. Thus, activated STAT6 alone is not sufficient to drive the GC B cell differentiation program. Consistent with the RNAseq data, we observed more IgE-and IgG1-expressing GC B cells and PCs as well as higher serum IgE and IgG1 levels in naïve CD19Cre STAT6vt as compared to control mice. Therefore, we conclude that constitutively active STAT6 does not modulate the population size of total GC B cells and PCs in naïve mice but promotes expression of IgE and IgG1.
Surprisingly, the enhanced IgE and IgG1 response was also observed after LCMV infection, which is characterized by a type 1 immune response with high levels of IFN-γ [24]. IFN-γ activates the STAT1 signaling pathway and inhibits CSR to IgE and IgG1 [25][26][27]. Using an LCMV-GP-specific ELISA, we could demonstrate that the IgE and IgG1 response is in part directed against the LCMV GP on the viral particle. On the other hand, we did not observe a reduction of the IgG2c response in LCMV-infected  CD19Cre STAT6vt mice, which suggests that activated STAT6 is not sufficient to suppress IgG2c CSR in B cells.
Taken together, we show here that selective expression of constitutively active STAT6 in mouse B cells leads to an enhanced IgE and IgG1 response that can even be directed against a viral pathogen. This suggests that chronic allergic inflammation with prolonged activation of the STAT6 signaling pathway in B cells might further be promoted by IgE responses to viral or bacterial antigens that would not occur under non-allergic conditions.

Mice
Rosa26 LSL-STAT6vt mice were generated by inserting a constitutively active version of STAT6 with a C-terminal FLAG-tag (STAT6vt [9]) behind a loxP-flanked Stop cassette into the genomic Rosa26 locus by standard ES cell technology using the pBigT/pROSA26-PA targeting vector system [28] and BALB/c-I ES cells [29]. Homologous recombination was identified by Southern blot analysis of EcoRI-digested genomic DNA. PCR with the following primer pair was used to identify the targeted allele in offspring mice: forward (5 -CACCATGTCTCTGTGGGGCCTAATTTCC-3 ) and reverse (5 -CAGCCCZGCTAGGCTCTCCTCAAAG-3 ). PCR conditions were 5 95°C, 35× (30 95°C, 30 58°C, 45 72°C), 5 72°C, and the size of the PCR product was 672 bp. Rosa26 LSL-STAT6vt mice (STAT6vt mice) were backcrossed to C57BL/6 background and then crossed with CD19Cre knock-in mice [13] to generate CD19Cre STAT6vt mice that are heterozygous for CD19Cre and STAT6vt. Heterozygous CD19Cre littermates served as controls. All mice were housed according to the institutional guidelines and both male and female mice were used at the age of 8-12 weeks. The animal experiments were approved by the Regierung von Unterfranken and performed in accordance with the German animal protection law.

LCMV infection
Mice were infected with 200 pfu of LCMV strain WE intravenously and analyzed at day 14 after infection.

Transcriptome analysis
Single cell suspension from spleen of naïve mice were generated by mechanical disruption. Erythrocytes were lysed with ACK-buffer C 2019 The Authors. European Journal of Immunology published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim (0.15 M NH 4 Cl, 1 mM KHO 3 , 0.1 mM Na 2 EDTA) and washed with FACS buffer (PBS, 2% FCS, 1 mg/mL NaN 3 ). Fc receptors were blocked with anti-mouse CD16/CD32 mAb and the cells stained at 4°C for 30 min with FITC-labeled CD19 (eBio 1D3, eBioscience) and PE Cy7-labeled CD3 (17A2, BioLegend). Immediately before sorting eBioscience TM Propidium Iodide Staining Solution (Thermo Fisher) was added to the cells. The cells were sorted into 1 mL FCS, pelleted and immediately lysed with RLTbuffer (RNeasy-Kit; Qiagen, Venlo, NL). The lysate was stored at -80°C until RNA isolation. RNA was isolated using the RNeasy-Kit (Qiagen). Purity and Concentration were determined via Nanodrop (Themofisher) and Qubit (Invitrogen; Thermo Fisher) and the RNA was sent to Novogene Co. (Hong-Kong) for transcriptome analysis (Ilumina platform).
For the bioinformatics analysis, log 2 transformed FPKM values were calculated and negative values were set back to zero before values were scaled and heatmaps were drawn with the gplots package in R (3.5.3; The R Foundation for Statistical Computing, Vienna, Austria). For gene set enrichment GSEA 3.0 software with FPKM values as input and recommended settings was used. For hallmark gene set analysis MSigDB database version 6.2 was employed using a build in function of the GSEA software to convert between mouse and human gene identifiers [32]. The raw data have been submitted to GEO database (accession no. GSE134408).

Statistical analysis
The sample size was chosen by preliminary observations. Statistical analysis was performed with SigmaPlot (12.3; Systat Software, San Jose, USA). Data are presented as mean with SE or SD, as indicated. Statistical significance was tested using an unpaired two-tailed Students t-test or Mann-Whitney U-test.