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. 2014 Mar 27;123(13):2108-15.
doi: 10.1182/blood-2013-10-533562. Epub 2014 Feb 14.

"VSports app下载" Increased BCR responsiveness in B cells from patients with chronic GVHD

Jessica L Allen  1 , Prasanthi V Tata, Matthew S Fore, Jenna Wooten, Sharmistha Rudra, Allison M Deal, Andrew Sharf, Todd Hoffert, Philip A Roehrs, Thomas C Shea, Jonathan S Serody, Kristy L Richards, Madan Jagasia, Stephanie J Lee, David Rizzieri, Mitchell E Horwitz, Nelson J Chao, Stefanie Sarantopoulos
Affiliations

Increased BCR responsiveness in B cells from patients with chronic GVHD

Jessica L Allen et al. Blood. .

Abstract (V体育平台登录)

Although B cells have emerged as important contributors to chronic graft-versus-host-disease (cGVHD) pathogenesis, the mechanisms responsible for their sustained activation remain unknown. We previously showed that patients with cGVHD have significantly increased B cell-activating factor (BAFF) levels and that their B cells are activated and resistant to apoptosis. Exogenous BAFF confers a state of immediate responsiveness to antigen stimulation in normal murine B cells VSports手机版. To address this in cGVHD, we studied B-cell receptor (BCR) responsiveness in 48 patients who were >1 year out from allogeneic hematopoietic stem cell transplantation (HSCT). We found that B cells from cGVHD patients had significantly increased proliferative responses to BCR stimulation along with elevated basal levels of the proximal BCR signaling components B cell linker protein (BLNK) and Syk. After initiation of BCR signaling, cGVHD B cells exhibited increased BLNK and Syk phosphorylation compared with B cells from patients without cGVHD. Blocking Syk kinase activity prevented relative post-HSCT BCR hyper-responsiveness of cGVHD B cells. These data suggest that a lowered BCR signaling threshold in cGVHD associates with increased B-cell proliferation and activation in response to antigen. We reveal a mechanism underpinning aberrant B-cell activation in cGVHD and suggest that therapeutic inhibition of the involved kinases may benefit these patients. .

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Figures

Figure 1
Figure 1
B cells from patients with cGVHD have increased BCR-driven proliferation. (A) B cells isolated from healthy donors and patients with and without cGVHD, stained with CFSE and stimulated with α-IgM (50 μg/mL) for 6 days. Unstimulated controls are shown as dashed lines. (Left) Representative histograms of B-cell proliferation from patients without cGVHD (gray area, thin line) and with cGVHD (white area, bold line). (Center) Frequency of B-cell proliferation quantified in healthy donors (HD, filled triangles), patients without cGVHD (−cGVHD, open circles), and patients with cGVHD (+cGVHD, filled squares). (Right) Proliferation index quantified in healthy donors (HD, filled triangles), patients without cGVHD (−cGVHD, open circles), and patients with cGVHD (+cGVHD, filled squares). Data are median ± range pooled from 4 independent experiments. *P < .05. (B) B cells isolated from patients with and without cGVHD, stained with CFSE, and stimulated with α-CD40 (100 ng/mL) + IL-4 (5 ng/mL) for 6 days. Unstimulated controls are shown as dashed lines. (Left) Representative histograms of B-cell proliferation from patients without cGVHD (gray area, thin line) and with cGVHD (white area, bold line). (Center) Frequency of B-cell proliferation quantified in patients without cGVHD (−cGVHD, open circles) and with cGVHD (+cGVHD, filled squares). (Right) Proliferation index quantified in patients without cGVHD (−cGVHD, open circles) and with cGVHD (+cGVHD, filled squares). Data are median ± range pooled from 3 independent experiments. *P < .05. Proliferation index = total number of divisions divided by the number of cells that went into division calculated using FlowJo Version 8.8.7.
Figure 2
Figure 2
Expression of BLNK and Syk is increased in cGVHD B cells ex vivo. Relative mRNA expression of 84 genes known to be involved in lymphocyte activation were analyzed in CD27+ and CD27− B cells from patients with cGVHD (n = 3) and healthy donors (n = 3). Data indicate the fold regulation of each gene that met statistical criteria, depicted in (A) CD27+ or (B) CD27− B cells from patients with cGVHD compared with healthy donors. Dashed lines indicate a greater than threefold increase (above line) or decrease (below line) in mRNA expression. Gray bars represent genes that are altered in both CD27+ and CD27− B-cell subsets. The P value for all genes is <.05; Student t test of replicate 2^(−ΔCt) values for each gene in the cGVHD group compared with healthy as calculated by SAbioscience Web-Based Array Analysis. Protein expression by mean fluorescence intensity (MFI) of (C) BLNK and (D) Syk in total peripheral B cells isolated from patients without cGVHD (−cGVHD, open circles) and with cGVHD (+cGVHD, filled squares). Data are median ± range from 2 independent experiments. *P <.05. (E) Protein expression by MFI of BLNK and Syk in B cells isolated from healthy donors (filled triangles), plated (1.5 × 106 cell/mL) and stimulated with α-IgM (50 μg/mL) or phosphate-buffered saline (PBS) as a control for 36 hours. Fold increase of α-IgM divided by PBS is depicted. *P = .003 (1-sample Student t test, theoretical mean = 1). Data are median ± range pooled from 2 independent experiments. *P < .05.
Figure 3
Figure 3
BCR-driven phosphorylation of BLNK is increased in CD27+ and CD27− B cells from patients with cGVHD. Phosphorylation of BLNK (tyrosine 84) and Syk (tyrosine 348) in CD27+ and CD27− B cells (CD20+) stimulated with α-IgM (5 μg/mL) or PBS as a control as indicated for 5 minutes. (A) (Left) representative dot plots of pBLNK in CD27+ B cells from a patient (upper) without cGVHD (−cGVHD) and (lower) with cGVHD (+cGVHD). Numbers indicate frequency of CD20+ CD27+ B cells that are pBLNK+. (Right) Frequency of BLNK phosphorylation in CD27+ B cells quantified in patients without cGVHD (n = 6, open circles) and with cGVHD (n = 5, filled squares). Data are median ± range pooled from 2 independent experiments, *P < .05. (B) (Left) Representative dot plots of pBLNK in CD27− B cells from a patient (upper) without cGVHD (−cGVHD) and (lower) with cGVHD (+cGVHD). Numbers indicate frequency of CD20+ CD27− B cells that are pBLNK+. (Right) Frequency of BLNK phosphorylation in CD27− B cells quantified in patients without cGVHD (n = 7, open circles) and with cGVHD (n = 6, filled squares). Data are median ± range pooled from 2 independent experiments, *P < .05. (C) (Left) Representative dot plots of pSyk in CD27+ B cells from a patient (upper) without cGVHD (−cGVHD) and (lower) with cGVHD (+cGVHD). Numbers indicate frequency of CD20+ CD27+ B cells that are pSyk+. (Right) Frequency of Syk phosphorylation in CD27+ B cells quantified in patients without cGVHD (n = 6, open circles) and with cGVHD (n = 5, filled squares). Data are median ± range pooled from 2 independent experiments, *P < .05. (D) (Left) Representative dot plots of pSyk in CD27− B cells from a patient (upper) without cGVHD (−cGVHD) and (lower) with cGVHD (+cGVHD). Numbers indicate frequency of CD20+ CD27− B cells that are pSyk+. (Right) Frequency of Syk phosphorylation in CD27− B cells quantified in patients without cGVHD (n = 7, open circles) and with cGVHD (n = 6, filled squares). Data are median ± range pooled from 2 independent experiments, *P < .05.
Figure 4
Figure 4
Surface expression of IgM is similar in CD27− and CD27+ B cells. Flow cytometric analysis of PBMCs for surface IgM expression. (A) representative histogram of CD27− B cells (CD19+ CD20+) from a patient without cGVHD (gray area, thin line) and with cGVHD (white area, bold line). Isotype is depicted as think gray line. (B) Representative histogram of CD27+ B cells (CD19+ CD20+) from a patient without cGVHD (gray area, thin line) and with cGVHD (white area, bold line). Gates indicate the frequency of B cells positive for IgM surface expression compared with isotype (thin gray line). (C) Frequency of IgM surface expression on CD27− and CD27+ B cells (CD19+ CD20+) quantified in patients without cGVHD (−cGVHD, n = 5, open circles) and with cGVHD (+cGVHD, n = 7 filled squares). Data are median ± range pooled from 2 independent experiments. *P < .05.
Figure 5
Figure 5
Kinase activity of Syk is important for BCR hyper-responsiveness in cGVHD B cells ex vivo. B cells isolated from patients without cGVHD (n = 4, open circles) and with cGVHD (n = 4, filled squares) stained with CFSE, treated with R406 (0, 0.01, and 0.1 μM) as indicated, and stimulated with (A-B) α-IgM (50 μg/mL) or (C-D) α-CD40 (100 ng/mL) + IL-4 (5 ng/mL) for 6 days. (A) Frequency of proliferation in response to α-IgM in B cells from patients (left) without cGVHD (open circles) and (right) with cGVHD (filled squares). (Left) ANOVA, P < .0001; *P < .05. (Right) ANOVA, P < .0067; *P < .05. (B) Percent change in number of B cells from A: [Percent change = (V2 – V1)/V1 × 100]. ANOVA, P = .0002; *P < .05. (C) Frequency of proliferation in response to α-CD40 + IL-4 in B cells from patients (left) without cGVHD (open circles) and (right) with cGVHD (filled squares). (D) Percent change in number of B cells from C: [Percent change = (V2 – V1)/V1 × 100]. Data are median ± range pooled from 3 independent experiments.
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