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Defective TLR9-driven STAT3 activation in B cells of patients with CVID
Accepted Manuscript Defective TLR9-driven STAT3 activation in B cells of patients with CVID
Arturo Borzutzky, Ingrid Rauter, Ari Fried, Rima Rachid, Douglas R. McDonald, Lennart Hammarstrom, Bodo Grimbacher, Roshini S. Abraham, Raif S. Geha PII: DOI: Reference:
S1521-6616(18)30498-4 doi:10.1016/j.clim.2018.08.008 YCLIM 8089
To appear in:
Clinical Immunology
Received date: Accepted date:
17 August 2018 17 August 2018
Please cite this article as: Arturo Borzutzky, Ingrid Rauter, Ari Fried, Rima Rachid, Douglas R. McDonald, Lennart Hammarstrom, Bodo Grimbacher, Roshini S. Abraham, Raif S. Geha , Defective TLR9-driven STAT3 activation in B cells of patients with CVID. Yclim (2018), doi:10.1016/j.clim.2018.08.008
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ACCEPTED MANUSCRIPT Defective TLR9-driven STAT3 activation in B cells of patients with CVID Arturo Borzutzky1#*, Ingrid Rauter1#, Ari Fried1, Rima Rachid1, Douglas R. McDonald1, Lennart Hammarstrom2, Bodo Grimbacher3, Roshini S. Abraham4, and Raif S. Geha1
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Division of Immunology, Children’s Hospital and Department of Pediatrics, Harvard Medical
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School, Boston, MA, 2Division of Clinical Immunology, Karolinska Institute, Huddinge,
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Sweden, 3Center of Chronic Immunodeficiency, Freiburg University Medical Center, Freiburg, Germany, and 4Department of Laboratory Medicine and Pathology, Mayo Clinic,
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Rochester, MN 55905 and Department of Pathology and Laboratory Medicine, Nationwide
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Children’s Hospital, Columbus, OH 43205.
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# Equal contributors
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* Current address: Departamento de Enfermedades Infecciosas e Inmunología Pediátrica,
Raif S. Geha, MD
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Corresponding author:
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Escuela de Medicina, Pontificia Universidad Católica de Chile, Santiago Chile
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James L. Gamble Professor of Pediatrics Harvard Medical School Chief, Division of Allergy/ Immunology/Rheumatology/ Dermatology Children's Hospital, Karp Building 10th floor-office#10-211 One Blackfan Circle, Boston, MA 02115 Tel: 617-919-2482 / Fax: 617-730-0528
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Declarations of interest: none
ACCEPTED MANUSCRIPT ABSTRACT B cell activation by Toll-like receptor 9 (TLR9) ligands is dependent on STAT3 and is important for optimal antibody responses to microbial antigens. B cells from patients with common variable immune deficiency (CVID) have impaired proliferation and differentiation in
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response to the TLR9 ligand CpG, despite normal levels of TLR9 expression. We
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demonstrate that CpG-driven STAT3 phosphorylation, but not activation of NFB and p38, is
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selectively impaired in B cells from CVID patients. These results suggest that defective
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STAT3 activation contributes to the defective TLR9 and antibody response of B cells in CVID.
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Keywords: common variable immunodeficiency, STAT3 Transcription Factor, B cells, Toll-
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like Receptor 9
ACCEPTED MANUSCRIPT Patients with CVID generally have normal numbers of circulating B cells. However, their B cell function is defective. Decreased numbers of switched IgD -CD27+ memory B cells and plasma cells are hallmarks of CVID [1-5]. The molecular mechanisms underlying CVID are largely unknown. A small number of CVID patients have homozygous mutations
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in genes that include those that encode for ICOS, BAFF-R, CD19, CD81, CD20, CD21
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NFB1, NFB2, CTLA4, BACH2, ADA2 and SEC61A1 and others [6]. Six to 10% of CVID
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patients have heterozygous mutations in the disease modifier gene TACI, but also rare homozygous TACI have been reported [7].
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B cells can be activated to secrete immunoglobulin by ligation of CD40, BCR, TACI,
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and Toll-like receptors (TLRs). B cells integrate signals from all four receptors to achieve optimal antibody production [8]. TLR9 binds unmethylated CpG oligodeoxynucleotides
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(ODN) motifs in bacterial DNA [9]. TLR9 engagement triggers B cell proliferation and IgG
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secretion, drives the differentiation of naive B cells into memory B cells, and promotes the generation of antibody-secreting plasma cells [10-12]. CpG drives STAT3 phosphorylation
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in B cells [13]. Importantly, the CpG response is impaired in B cells from STAT3 deficient
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patients and B cells from DOCK8 deficient patients, which have defective CpG-driven STAT3 phosphorylation, impaired antibody formation and decreased memory B cells [13].
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CpG-driven IgG production and generation of memory B cells are defective in CVID patients [12, 14, 15]. We herein demonstrate that CpG-driven phosphorylation of STAT3 is defective in B cells from CVID patients with and without TACI mutation. We studied ten adult patients who fulfilled the clinical and laboratory diagnostic criteria for CVID [6] for response to CpG-driven proliferation, IgG secretion in PBMCs, IgG secretion in purified B cell subpopulations and STAT3-driven phosphorylation in PBMCs
ACCEPTED MANUSCRIPT and purified B cells. All were on immunoglobulin replacement therapy. Four had a heterozygous mutation in TACI: p.C104R in three and p.A181R in one. All patients had normal surface expression of CD19, CD20 CD21 and ICOS on B cells by FACS, The mean percentage of CD19+ B cells in PBMCs was comparable to that of adult controls (Fig. 1A).
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The mean percentage of CD19+IgD-CD27+ switched memory B cells was significantly
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diminished in the patients (Fig. 1A). Proliferation and IgG secretion by the patients’ PBMCs
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in response to stimulation with CpG were significantly lower compared to controls (Fig. 1B). In contrast, PBMCs from the CVID patients proliferated and secreted IgG normally in
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response to anti-CD40+IL-4 (Supplementary Fig. 1) [16-18]. Study of an additional cohort
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of 12 CVID patients that included three with the TACI p.C104R mutation and seven with the TACI p.A181E mutation revealed similar results with proliferation and IgG secretion that
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were significantly impaired in response to CpG stimulation, but preserved in response to
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anti-CD40+IL-4 stimulation, compared to 11 healthy controls (data not shown). These results indicate that, in agreement with previous studies [12, 14, 15], TLR9-driven B cell
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activation is selectively impaired in our CVID patients.
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Lymph nodes from patients with CVID lack plasmablasts, but B cells from these LNs may express normal amounts of the transcription factor BLIMP1 required for plasma cell
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differentiation [5, 19-21]. CpG-driven generation of CD27hiCD38hi plasmablasts was significantly reduced in the patients (Fig. 1C); however, CpG induced expression of BLIMP1 mRNA in B cells was normal (Supplementary Fig. 2); thus, factors other than BLIMP1 are responsible for the defective plasmablast differentiation. A subset of B cells expresses CD70 [22]. Interaction of CD70 and CD27 promotes B cell differentiation [17, 23, 24]. To examine whether defective CD27 expression underlies
ACCEPTED MANUSCRIPT the impaired response of CVID B cells to CpG, we purified CD19+ B cells from patients with CVID and controls by negative selection, sorted them into CD27 + and CD27- B cells and examined their response to CpG. The purity of the populations was >95%. Because of the large amounts of blood needed, we could study only three CVID patients (one with no TACI
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mutation, one with p.C104R TACI and one with p.A181E TACI) and three controls. Purified
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CD19+ B cells from CVID patients secreted significantly less IgG in response to CpG than
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those from normal controls (Fig. 1D), confirming previous observations that the impaired CpG-driven IgG secretion is B cell intrinsic [12]. Consistent with other reports [25, 26], CpG
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stimulation induced IgG secretion in both CD27- naïve B cells and CD27+ memory B cells
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from normal controls, but both CD27- naïve and CD27+ memory B cell populations from CVID patients secreted significantly less IgG in response to CpG stimulation than their
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normal counterparts (Fig. 1D).
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Impaired CpG-driven proliferation and IgG secretion in B cells from CVID patients could have been due to reduced TLR9 expression and/or defective TLR9 signaling. In
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agreement with a previous report [12], TLR9 mRNA expression was comparable in CD19+ B
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cells from CVID patients and controls (data not shown). CpG stimulation of B cells causes NFB activation by the classical pathway, which depends on the phosphorylation and
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degradation of IB. It also results in phosphorylation of p38 and STAT3 [27]. CpG stimulation caused rapid degradation of IB and phosphorylation of p38 in purified B cells that were comparable in patients and controls (Fig. 2A,B). CpG stimulation caused delayed phosphorylation of STAT3 in PBMCs and purified B cells from normal controls that became evident after 3 hrs, and increased further after 4 hrs (Fig. 2C,D). In contrast CpG-driven STAT3 phosphorylation was significantly impaired in PBMCs as well as in purified B cells
ACCEPTED MANUSCRIPT from CVID patients (Fig. 2C,D). Interferon- stimulation caused comparable phosphorylation of STAT3 in B cells from CVID patients and controls (Fig. 2D). Furthermore, STAT3 phosphorylation in response to IL-6 and IL-21 stimulation was comparable in EBV transformed B cell lines from CVID patients and controls (Fig. 2E), consistent with normal
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IgG secretion by B cells of CVID patients in response to stimulation with anti-CD40+IL-21
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[18, 28]. These results indicate that the defect in STAT3 phosphorylation in CVID B cells
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was selective to CpG stimulation.
in
B
cells
of
CVID
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A recent study reported increased STAT3 phosphorylation
patients stimulated for 24 hrs with CpG, as assessed by intracellular flow cytometry [29].
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However, the increase in pSTAT3 was accompanied by an increase in STAT3 protein expression, and appears limited to CD27- B cells. The discrepancy with our results may
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relate to differences in techniques and/or patient populations. CpG stimulation causes B
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cells to produce IL-6 and IL-10 [30]. Production of these two cytokines by CpG stimulated B cells was reported to be impaired in CVID patients [12, 14]. Addition of rIL-6 or rIL-10 to
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CpG stimulated cultures of PBMCs from CVID patients did not correct their defective IgG
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secretion (data not shown). Thus, impaired secretion of IL-6 and IL-10 by B cells is unlikely to explain, at least by itself, the defective response of B cells to CpG in CVID.
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In summary, we demonstrate that CpG-driven STAT3 phosphorylation in B cells is selectively impaired in seven CVID patients studied. This defect may contribute to the defective antibody response at least in some of the patients with CVID.
ACCEPTED MANUSCRIPT MATERIALS & METHODS Patients and controls All CVID patients had history of recurrent sinopulmonary infections, and all fulfilled the diagnostic criteria for CVID [31]. Serum IgG was reduced in all, serum
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IgA in 8, and serum IgM in 6. None of the subjects had an acute infection at the
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time of study. Informed consent was obtained from all subjects.
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Antibodies and flow cytometry
Single cell suspensions were stained with fluorochrome-conjugated
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antibodies in PBS containing 0.5% BSA, washed, and analyzed on a FACS
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Canto instrument (Becton Dickinson). Conjugated anti-human antibodies were: CD19-FITC (eBioscience), IgD-FITC and CD27-PE (Invitrogen), CD45-PerCP,
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CD19-APC and CD38-PECy7 (BD-Pharmingen) and the corresponding isotype
CD27 and CD38.
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Cell isolation
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controls. Plasmablasts were identified by four colour staining for CD45, CD19,
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PBMCs were isolated from heparinized blood by density-gradient centrifugation on Ficoll Hypaque (Amersham Pharmacia Biotech). Cells were
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washed and resuspended in complete RPMI-1640 medium, containing 10% heatinactivated
FCS
(Hyclone
Laboratories),
2
mM
L-glutamine,
50g/mL
streptomycin, and 100 U/mL penicillin (Life Technologies Inc.). Purified B cells were isolated from PBMCs by negative selection for CD19+ cells and memory B versus naïve B cells were isolated cells from purified CD19+ cells for CD27+ cells
ACCEPTED MANUSCRIPT using the Memory B Cell Isolation Kit (Miltenyi). FACS analysis of the purified cell populations showed more than 90% purity. Cell cultures for proliferation and immunoglobulin production in vitro For proliferation assays, PBMCs were cultured in complete medium
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(1.0x106 cells/mL) alone or in the presence of CpG ODN2006 (0.075 M,
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Invivogen), or anti-CD40 mAb 626 (5 g/mL, a kind gift of S.M. Fu, University of
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Virginia, Charlottesville, Virgina, USA) plus IL-4 (5 ng/mL, R&D) for 72 hours, pulsed with 1 Ci 3H-thymidine for an additional 16 hours, then harvested and
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scintillation counted. For immunoglobulin production PBMCs and purified B cells
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(1.5 x 106 cells/mL) were cultured in complete medium for 14 days, then supernatants were harvested and assessed for their immunoglobulin contents by
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ELISA as previously described [32]. Net IgG and IgE synthesis were calculated
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by substracting the amount of IgG or IgE in unstimulated cultures. Quantitative real time PCR
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RNA from PBMCs was prepared using Trizol (Invitrogen) and transcribed
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into cDNA using Superscript One Step RT-PCR kit (Invitrogen). Real-time PCR reactions were run on cDNA by using ABI Prism 7300 (Applied Biosystems).
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Taqman primers with 6-carboxyfluorescein labeled probes for BLIMP-1 and the housekeeping gene GADPH were obtained from Applied Biosystems. Relative expression was determined by using the method described by Pfaffl [33]. TLR9-mediated signaling in PBMCs and purified B cells PBMCs or purified B cells (1 x106 cells/condition) were stimulated in a volume of 200 μL medium with CpG DNA ODN2006 (2.5 μM/L, Invivogen) or
ACCEPTED MANUSCRIPT IFN-α (1000 U/ml; Biosource). EBV-B cells from were stimulated with rIL-6 (2 ng/ml, R&D Systems), or rIL-21 (10 ng/ml, Cell signaling). After stimulation, cells were lysed in Sample Buffer (62.5 mM TRIS, pH 6.8, 2% wt/vol SDS, 10% glycerol, 2% -mercaptoethanol, 0.01% bromophenol blue). Proteins were
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resolved by 10% SDS-PAGE (BioRad) and transferred to nitrocellulose
STAT3, phospho-p38, and p38 antibodies (all from
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phospho-STAT3 antibody
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membranes (Invitrogen). Membranes were probed with the following antibodies:
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Cell Signaling), IBantibody (Santa Cruz Biotechnology), and actin monoclonal antibody (Chemicon International). Western blotting was performed according to
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the manufacturer’s recommendations. Statistical analysis
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Two-tailed Student's t-test was used to compare the differences between
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groups using the GraphPad PRISM software (GraphPad Software Inc.). P-values
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<0.05 were considered significant.
ACCEPTED MANUSCRIPT ACKNOWLEDGMENTS. This work was supported by NIH grants AI-076210 and T32-AI-007512, March of Dimes grant #6-FY07-285 and Austrian Science Fund J2744-B12 (I.R.).
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We thank Dr. Manuel Leyva-Castillo for help in preparing the figures.
ACCEPTED MANUSCRIPT FIGURE LEGENDS Fig. 1.
Impaired CpG response of B cells from CVID patients. A.
Percentages of CD19+ B cells (left) and IgD-CD27+ switched memory B cells (right). B. Proliferation (left), and IgG production (right) by PBMCs to CpG (0.075 Values for proliferation and IgG secretion represent net values, obtained by
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substracting the values of unstimulated cultures. C. Representative FACS
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analysis of CD27 and CD38 expression by CD19+ gated cells (left) and percentages of CD27hiCD38hi plasmablasts (right). D. IgG production by purified
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CD19+ B cells, naïve and memory B cells stimulated with CpG. Color of circles
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for CVID patients in A, B and D represent TACI mutation: black: none, blue: C104R red/Purple: A181E. Bars in A, B and D represent means. Columns and
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bars in C represent mean and S.D. (n = 7 controls and 8 patients). HC: healthy
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control. *p<0.05, **p<0.01, ***p<0.001.
Fig. 2. Defective CpG driven STAT3 phosphorylation in purified B
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cells from CVID patients.
A, B. Representative Western blot of IB
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degradation (A) and p38 phosphorylation (B) following CpG stimulation. Similar results were obtained in two other patients studied. C, D. Phosphorylation of and
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STAT3 in PMBCs (C) and purified B cells (D) in response to stimulation with CpG. In B cells stimulation with IFN- was used as a control. The left panels show a representative experiment. The plots depict the results from PMBCs of 7 patients and controls in C, and from B cells from 3 patients and 3 controls studied in D. Results are expressed as a ratio of the intensity of the pSTAT3 band over the B-cell Linker Protein (BLNK) and STAT3 bands in C and D respectively, as
ACCEPTED MANUSCRIPT assessed by scanning densitometry 4 hrs post-stimulation with CpG and 3 hrs after stimulation with IFN-. Similar results were obtained after CpG stimulation for 3 hrs. E. Phosphorylation of STAT3 in EBV-B cells following stimulation for 10 min with IL-6 and IL-21. Similar results were obtained in two other patients
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studied. Bars represent means. HC: healthy control. *p<0.05, ** p<0.01. n.s.=not
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significant.
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ACCEPTED MANUSCRIPT SUPPLEMENTAL FUGURES Fig. S1. Normal ligation. A, B.
response of B cells from CVID patients to CD40
Proliferation and IgG production by PBMCs to anti-CD40+IL-4.
Values for proliferation and IgG secretion represent net values, obtained by
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substracting the values of unstimulated cultures. Color of circles for CVID
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patients represent TACI mutation: black: none, red:C104R, blue: A181E.
Fig. S2.
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Horizontal lines represent means.
BLIMP1 expression in CVID patients and controls
qPCR
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analysis of BLIMP-1 mRNA expression relative to GAPDH mRNA in unstimulated
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and CpG stimulated PBMCs. Horizontal lines represent means. ** p <0.01, *** p
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<0.001, n.s.=not significant.
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Highlights CVID patients’ B cells have impaired CpG-induced IgG production and proliferation CpG-driven STAT3 phosphorylation is impaired in B cells from CVID patients STAT3 phosphorylation induced by IL-6, IL-21 and IFN- is preserved. Defective CpG-driven STAT3 phosphorylation may contribute to the CVID phenotype.
Figure 1
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Arturo Borzutzky, Ingrid Rauter, Ari Fried, Rima Rachid, Douglas R. McDonald, Lennart Hammarstrom, Bodo Grimbacher, Roshini S. Abraham, Raif S. Geha PII: DOI: Reference:
S1521-6616(18)30498-4 doi:10.1016/j.clim.2018.08.008 YCLIM 8089
To appear in:
Clinical Immunology
Received date: Accepted date:
17 August 2018 17 August 2018
Please cite this article as: Arturo Borzutzky, Ingrid Rauter, Ari Fried, Rima Rachid, Douglas R. McDonald, Lennart Hammarstrom, Bodo Grimbacher, Roshini S. Abraham, Raif S. Geha , Defective TLR9-driven STAT3 activation in B cells of patients with CVID. Yclim (2018), doi:10.1016/j.clim.2018.08.008
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ACCEPTED MANUSCRIPT Defective TLR9-driven STAT3 activation in B cells of patients with CVID Arturo Borzutzky1#*, Ingrid Rauter1#, Ari Fried1, Rima Rachid1, Douglas R. McDonald1, Lennart Hammarstrom2, Bodo Grimbacher3, Roshini S. Abraham4, and Raif S. Geha1
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Division of Immunology, Children’s Hospital and Department of Pediatrics, Harvard Medical
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School, Boston, MA, 2Division of Clinical Immunology, Karolinska Institute, Huddinge,
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Sweden, 3Center of Chronic Immunodeficiency, Freiburg University Medical Center, Freiburg, Germany, and 4Department of Laboratory Medicine and Pathology, Mayo Clinic,
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Rochester, MN 55905 and Department of Pathology and Laboratory Medicine, Nationwide
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Children’s Hospital, Columbus, OH 43205.
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# Equal contributors
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* Current address: Departamento de Enfermedades Infecciosas e Inmunología Pediátrica,
Raif S. Geha, MD
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Corresponding author:
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Escuela de Medicina, Pontificia Universidad Católica de Chile, Santiago Chile
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James L. Gamble Professor of Pediatrics Harvard Medical School Chief, Division of Allergy/ Immunology/Rheumatology/ Dermatology Children's Hospital, Karp Building 10th floor-office#10-211 One Blackfan Circle, Boston, MA 02115 Tel: 617-919-2482 / Fax: 617-730-0528
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Declarations of interest: none
ACCEPTED MANUSCRIPT ABSTRACT B cell activation by Toll-like receptor 9 (TLR9) ligands is dependent on STAT3 and is important for optimal antibody responses to microbial antigens. B cells from patients with common variable immune deficiency (CVID) have impaired proliferation and differentiation in
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response to the TLR9 ligand CpG, despite normal levels of TLR9 expression. We
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demonstrate that CpG-driven STAT3 phosphorylation, but not activation of NFB and p38, is
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selectively impaired in B cells from CVID patients. These results suggest that defective
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STAT3 activation contributes to the defective TLR9 and antibody response of B cells in CVID.
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Keywords: common variable immunodeficiency, STAT3 Transcription Factor, B cells, Toll-
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like Receptor 9
ACCEPTED MANUSCRIPT Patients with CVID generally have normal numbers of circulating B cells. However, their B cell function is defective. Decreased numbers of switched IgD -CD27+ memory B cells and plasma cells are hallmarks of CVID [1-5]. The molecular mechanisms underlying CVID are largely unknown. A small number of CVID patients have homozygous mutations
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in genes that include those that encode for ICOS, BAFF-R, CD19, CD81, CD20, CD21
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NFB1, NFB2, CTLA4, BACH2, ADA2 and SEC61A1 and others [6]. Six to 10% of CVID
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patients have heterozygous mutations in the disease modifier gene TACI, but also rare homozygous TACI have been reported [7].
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B cells can be activated to secrete immunoglobulin by ligation of CD40, BCR, TACI,
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and Toll-like receptors (TLRs). B cells integrate signals from all four receptors to achieve optimal antibody production [8]. TLR9 binds unmethylated CpG oligodeoxynucleotides
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(ODN) motifs in bacterial DNA [9]. TLR9 engagement triggers B cell proliferation and IgG
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secretion, drives the differentiation of naive B cells into memory B cells, and promotes the generation of antibody-secreting plasma cells [10-12]. CpG drives STAT3 phosphorylation
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in B cells [13]. Importantly, the CpG response is impaired in B cells from STAT3 deficient
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patients and B cells from DOCK8 deficient patients, which have defective CpG-driven STAT3 phosphorylation, impaired antibody formation and decreased memory B cells [13].
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CpG-driven IgG production and generation of memory B cells are defective in CVID patients [12, 14, 15]. We herein demonstrate that CpG-driven phosphorylation of STAT3 is defective in B cells from CVID patients with and without TACI mutation. We studied ten adult patients who fulfilled the clinical and laboratory diagnostic criteria for CVID [6] for response to CpG-driven proliferation, IgG secretion in PBMCs, IgG secretion in purified B cell subpopulations and STAT3-driven phosphorylation in PBMCs
ACCEPTED MANUSCRIPT and purified B cells. All were on immunoglobulin replacement therapy. Four had a heterozygous mutation in TACI: p.C104R in three and p.A181R in one. All patients had normal surface expression of CD19, CD20 CD21 and ICOS on B cells by FACS, The mean percentage of CD19+ B cells in PBMCs was comparable to that of adult controls (Fig. 1A).
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The mean percentage of CD19+IgD-CD27+ switched memory B cells was significantly
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diminished in the patients (Fig. 1A). Proliferation and IgG secretion by the patients’ PBMCs
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in response to stimulation with CpG were significantly lower compared to controls (Fig. 1B). In contrast, PBMCs from the CVID patients proliferated and secreted IgG normally in
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response to anti-CD40+IL-4 (Supplementary Fig. 1) [16-18]. Study of an additional cohort
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of 12 CVID patients that included three with the TACI p.C104R mutation and seven with the TACI p.A181E mutation revealed similar results with proliferation and IgG secretion that
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were significantly impaired in response to CpG stimulation, but preserved in response to
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anti-CD40+IL-4 stimulation, compared to 11 healthy controls (data not shown). These results indicate that, in agreement with previous studies [12, 14, 15], TLR9-driven B cell
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activation is selectively impaired in our CVID patients.
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Lymph nodes from patients with CVID lack plasmablasts, but B cells from these LNs may express normal amounts of the transcription factor BLIMP1 required for plasma cell
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differentiation [5, 19-21]. CpG-driven generation of CD27hiCD38hi plasmablasts was significantly reduced in the patients (Fig. 1C); however, CpG induced expression of BLIMP1 mRNA in B cells was normal (Supplementary Fig. 2); thus, factors other than BLIMP1 are responsible for the defective plasmablast differentiation. A subset of B cells expresses CD70 [22]. Interaction of CD70 and CD27 promotes B cell differentiation [17, 23, 24]. To examine whether defective CD27 expression underlies
ACCEPTED MANUSCRIPT the impaired response of CVID B cells to CpG, we purified CD19+ B cells from patients with CVID and controls by negative selection, sorted them into CD27 + and CD27- B cells and examined their response to CpG. The purity of the populations was >95%. Because of the large amounts of blood needed, we could study only three CVID patients (one with no TACI
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mutation, one with p.C104R TACI and one with p.A181E TACI) and three controls. Purified
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CD19+ B cells from CVID patients secreted significantly less IgG in response to CpG than
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those from normal controls (Fig. 1D), confirming previous observations that the impaired CpG-driven IgG secretion is B cell intrinsic [12]. Consistent with other reports [25, 26], CpG
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stimulation induced IgG secretion in both CD27- naïve B cells and CD27+ memory B cells
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from normal controls, but both CD27- naïve and CD27+ memory B cell populations from CVID patients secreted significantly less IgG in response to CpG stimulation than their
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normal counterparts (Fig. 1D).
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Impaired CpG-driven proliferation and IgG secretion in B cells from CVID patients could have been due to reduced TLR9 expression and/or defective TLR9 signaling. In
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agreement with a previous report [12], TLR9 mRNA expression was comparable in CD19+ B
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cells from CVID patients and controls (data not shown). CpG stimulation of B cells causes NFB activation by the classical pathway, which depends on the phosphorylation and
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degradation of IB. It also results in phosphorylation of p38 and STAT3 [27]. CpG stimulation caused rapid degradation of IB and phosphorylation of p38 in purified B cells that were comparable in patients and controls (Fig. 2A,B). CpG stimulation caused delayed phosphorylation of STAT3 in PBMCs and purified B cells from normal controls that became evident after 3 hrs, and increased further after 4 hrs (Fig. 2C,D). In contrast CpG-driven STAT3 phosphorylation was significantly impaired in PBMCs as well as in purified B cells
ACCEPTED MANUSCRIPT from CVID patients (Fig. 2C,D). Interferon- stimulation caused comparable phosphorylation of STAT3 in B cells from CVID patients and controls (Fig. 2D). Furthermore, STAT3 phosphorylation in response to IL-6 and IL-21 stimulation was comparable in EBV transformed B cell lines from CVID patients and controls (Fig. 2E), consistent with normal
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IgG secretion by B cells of CVID patients in response to stimulation with anti-CD40+IL-21
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[18, 28]. These results indicate that the defect in STAT3 phosphorylation in CVID B cells
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was selective to CpG stimulation.
in
B
cells
of
CVID
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A recent study reported increased STAT3 phosphorylation
patients stimulated for 24 hrs with CpG, as assessed by intracellular flow cytometry [29].
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However, the increase in pSTAT3 was accompanied by an increase in STAT3 protein expression, and appears limited to CD27- B cells. The discrepancy with our results may
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relate to differences in techniques and/or patient populations. CpG stimulation causes B
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cells to produce IL-6 and IL-10 [30]. Production of these two cytokines by CpG stimulated B cells was reported to be impaired in CVID patients [12, 14]. Addition of rIL-6 or rIL-10 to
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CpG stimulated cultures of PBMCs from CVID patients did not correct their defective IgG
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secretion (data not shown). Thus, impaired secretion of IL-6 and IL-10 by B cells is unlikely to explain, at least by itself, the defective response of B cells to CpG in CVID.
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In summary, we demonstrate that CpG-driven STAT3 phosphorylation in B cells is selectively impaired in seven CVID patients studied. This defect may contribute to the defective antibody response at least in some of the patients with CVID.
ACCEPTED MANUSCRIPT MATERIALS & METHODS Patients and controls All CVID patients had history of recurrent sinopulmonary infections, and all fulfilled the diagnostic criteria for CVID [31]. Serum IgG was reduced in all, serum
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IgA in 8, and serum IgM in 6. None of the subjects had an acute infection at the
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time of study. Informed consent was obtained from all subjects.
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Antibodies and flow cytometry
Single cell suspensions were stained with fluorochrome-conjugated
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antibodies in PBS containing 0.5% BSA, washed, and analyzed on a FACS
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Canto instrument (Becton Dickinson). Conjugated anti-human antibodies were: CD19-FITC (eBioscience), IgD-FITC and CD27-PE (Invitrogen), CD45-PerCP,
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CD19-APC and CD38-PECy7 (BD-Pharmingen) and the corresponding isotype
CD27 and CD38.
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Cell isolation
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controls. Plasmablasts were identified by four colour staining for CD45, CD19,
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PBMCs were isolated from heparinized blood by density-gradient centrifugation on Ficoll Hypaque (Amersham Pharmacia Biotech). Cells were
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washed and resuspended in complete RPMI-1640 medium, containing 10% heatinactivated
FCS
(Hyclone
Laboratories),
2
mM
L-glutamine,
50g/mL
streptomycin, and 100 U/mL penicillin (Life Technologies Inc.). Purified B cells were isolated from PBMCs by negative selection for CD19+ cells and memory B versus naïve B cells were isolated cells from purified CD19+ cells for CD27+ cells
ACCEPTED MANUSCRIPT using the Memory B Cell Isolation Kit (Miltenyi). FACS analysis of the purified cell populations showed more than 90% purity. Cell cultures for proliferation and immunoglobulin production in vitro For proliferation assays, PBMCs were cultured in complete medium
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(1.0x106 cells/mL) alone or in the presence of CpG ODN2006 (0.075 M,
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Invivogen), or anti-CD40 mAb 626 (5 g/mL, a kind gift of S.M. Fu, University of
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Virginia, Charlottesville, Virgina, USA) plus IL-4 (5 ng/mL, R&D) for 72 hours, pulsed with 1 Ci 3H-thymidine for an additional 16 hours, then harvested and
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scintillation counted. For immunoglobulin production PBMCs and purified B cells
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(1.5 x 106 cells/mL) were cultured in complete medium for 14 days, then supernatants were harvested and assessed for their immunoglobulin contents by
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ELISA as previously described [32]. Net IgG and IgE synthesis were calculated
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by substracting the amount of IgG or IgE in unstimulated cultures. Quantitative real time PCR
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RNA from PBMCs was prepared using Trizol (Invitrogen) and transcribed
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into cDNA using Superscript One Step RT-PCR kit (Invitrogen). Real-time PCR reactions were run on cDNA by using ABI Prism 7300 (Applied Biosystems).
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Taqman primers with 6-carboxyfluorescein labeled probes for BLIMP-1 and the housekeeping gene GADPH were obtained from Applied Biosystems. Relative expression was determined by using the method described by Pfaffl [33]. TLR9-mediated signaling in PBMCs and purified B cells PBMCs or purified B cells (1 x106 cells/condition) were stimulated in a volume of 200 μL medium with CpG DNA ODN2006 (2.5 μM/L, Invivogen) or
ACCEPTED MANUSCRIPT IFN-α (1000 U/ml; Biosource). EBV-B cells from were stimulated with rIL-6 (2 ng/ml, R&D Systems), or rIL-21 (10 ng/ml, Cell signaling). After stimulation, cells were lysed in Sample Buffer (62.5 mM TRIS, pH 6.8, 2% wt/vol SDS, 10% glycerol, 2% -mercaptoethanol, 0.01% bromophenol blue). Proteins were
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resolved by 10% SDS-PAGE (BioRad) and transferred to nitrocellulose
STAT3, phospho-p38, and p38 antibodies (all from
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phospho-STAT3 antibody
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membranes (Invitrogen). Membranes were probed with the following antibodies:
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Cell Signaling), IBantibody (Santa Cruz Biotechnology), and actin monoclonal antibody (Chemicon International). Western blotting was performed according to
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the manufacturer’s recommendations. Statistical analysis
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Two-tailed Student's t-test was used to compare the differences between
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groups using the GraphPad PRISM software (GraphPad Software Inc.). P-values
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<0.05 were considered significant.
ACCEPTED MANUSCRIPT ACKNOWLEDGMENTS. This work was supported by NIH grants AI-076210 and T32-AI-007512, March of Dimes grant #6-FY07-285 and Austrian Science Fund J2744-B12 (I.R.).
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We thank Dr. Manuel Leyva-Castillo for help in preparing the figures.
ACCEPTED MANUSCRIPT FIGURE LEGENDS Fig. 1.
Impaired CpG response of B cells from CVID patients. A.
Percentages of CD19+ B cells (left) and IgD-CD27+ switched memory B cells (right). B. Proliferation (left), and IgG production (right) by PBMCs to CpG (0.075 Values for proliferation and IgG secretion represent net values, obtained by
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substracting the values of unstimulated cultures. C. Representative FACS
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analysis of CD27 and CD38 expression by CD19+ gated cells (left) and percentages of CD27hiCD38hi plasmablasts (right). D. IgG production by purified
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CD19+ B cells, naïve and memory B cells stimulated with CpG. Color of circles
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for CVID patients in A, B and D represent TACI mutation: black: none, blue: C104R red/Purple: A181E. Bars in A, B and D represent means. Columns and
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bars in C represent mean and S.D. (n = 7 controls and 8 patients). HC: healthy
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control. *p<0.05, **p<0.01, ***p<0.001.
Fig. 2. Defective CpG driven STAT3 phosphorylation in purified B
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cells from CVID patients.
A, B. Representative Western blot of IB
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degradation (A) and p38 phosphorylation (B) following CpG stimulation. Similar results were obtained in two other patients studied. C, D. Phosphorylation of and
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STAT3 in PMBCs (C) and purified B cells (D) in response to stimulation with CpG. In B cells stimulation with IFN- was used as a control. The left panels show a representative experiment. The plots depict the results from PMBCs of 7 patients and controls in C, and from B cells from 3 patients and 3 controls studied in D. Results are expressed as a ratio of the intensity of the pSTAT3 band over the B-cell Linker Protein (BLNK) and STAT3 bands in C and D respectively, as
ACCEPTED MANUSCRIPT assessed by scanning densitometry 4 hrs post-stimulation with CpG and 3 hrs after stimulation with IFN-. Similar results were obtained after CpG stimulation for 3 hrs. E. Phosphorylation of STAT3 in EBV-B cells following stimulation for 10 min with IL-6 and IL-21. Similar results were obtained in two other patients
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studied. Bars represent means. HC: healthy control. *p<0.05, ** p<0.01. n.s.=not
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significant.
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C4b-binding protein (C4BP) activates B cells through the CD40 receptor,
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Immunity, 18 (2003) 837-848.
[33] M.W. Pfaffl, A new mathematical model for relative quantification in real-time
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RT-PCR, Nucleic acids research, 29 (2001) e45.
ACCEPTED MANUSCRIPT SUPPLEMENTAL FUGURES Fig. S1. Normal ligation. A, B.
response of B cells from CVID patients to CD40
Proliferation and IgG production by PBMCs to anti-CD40+IL-4.
Values for proliferation and IgG secretion represent net values, obtained by
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substracting the values of unstimulated cultures. Color of circles for CVID
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patients represent TACI mutation: black: none, red:C104R, blue: A181E.
Fig. S2.
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Horizontal lines represent means.
BLIMP1 expression in CVID patients and controls
qPCR
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analysis of BLIMP-1 mRNA expression relative to GAPDH mRNA in unstimulated
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and CpG stimulated PBMCs. Horizontal lines represent means. ** p <0.01, *** p
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<0.001, n.s.=not significant.
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Highlights CVID patients’ B cells have impaired CpG-induced IgG production and proliferation CpG-driven STAT3 phosphorylation is impaired in B cells from CVID patients STAT3 phosphorylation induced by IL-6, IL-21 and IFN- is preserved. Defective CpG-driven STAT3 phosphorylation may contribute to the CVID phenotype.
Figure 1
Figure 2