49 Increased Blnk and Syk in B Cells From Chronic Graft-Versus-Host Disease Patients: Identification of Novel Therapeutic Targets

Track: BMT Tandem "Scientific" Meeting
Thursday, February 14, 2013, 4:45 PM-6:15 PM
Ballroom E-H (Salt Palace Convention Center)
Jessica L Allen, PhD , Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, NC
George Fedoriw, MD , Department of Pathology and Laboratory Medicine, University of North Carolina School of Medicine, Chapel Hill, NC
Jenna Wooten, PhD , Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, NC
Matthew Fore , Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, NC
Philip A Roehrs, MD , Division of Pediatric Hematology-Oncology and Bone Marrow and Stem Cell Transplant Program, University of North Carolina School of Medicine, Chapel Hill, NC
Paul Armistead, MD, PhD , Hematology / Oncology, Univeristy of North Carolina, Chapel Hill, NC
James Coghill, MD , Divison of Hematology-Oncology, Bone Marrow and Stem Cell Transplant Program, University of North Carolina School of Medicine, Chapel Hill, NC
Thomas C. Shea, MD , Bone Marrow Transplant Program, Division of Medical Oncology, University of North Carolina at Chapel Hill, Chapel Hill, NC
Kristy Richards, MD, PhD , Lineberger Comprehensive Cancer Center and the Institute for Pharmacogenomics and Individualized Therapy, University of North Carolina, Chapel Hill, NC
Stephanie J. Lee, MD, MPH , Clinical Transplant Research, Fred Hutchinson Cancer Research Center, Seattle, WA
Krista Rowe, RN, MSN, AOCNS , Division of Hematologic Malignancies and Cellular Therapy, Duke University Health System, Durham, NC
David Rizzieri, M.D. , Duke Universtiy Medical Center, Durham, NC
Nelson J. Chao, MD , Duke University Medical Center, Durham, NC
Jonathan S. Serody, MD , Comprehensive Cancer Center, University of North Carolina, Chapel Hill, NC
Stefanie Sarantopoulos, MD, PhD , Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, NC

In previous work, we found that CD27+ B cells from patients with chronic GVHD (cGVHD) are metabolically active and capable of constitutive IgG production via BAFF-associated pathways. As B Cell Receptor (BCR) signaling is vital for B cell survival and activation, we hypothesized that antigen engagement of BCR confers distinct signaling in potentially pathologic CD27+ B cells in human cGVHD. We first aimed to identify candidate genes using pathway-focused mRNA expression profiling of highly purified CD27+ and CD27- B cell populations from 3 patients with cGVHD and 3 healthy individuals. A unique array signature was identified, confirming that circulating CD27+ B cells in cGVHD are distinct from healthy 'memory' B cells. Of the 89 genes examined, 3 transcripts were significantly increased in CD27+ B cells from patients with cGVHD compared to healthy individuals. B-cell linker protein (BLNK), downstream of BCR ligation, was significantly increased 5.5 fold (p=0.01) in CD27+ B cells from patients with cGVHD compared to CD27+ B cells in healthy individuals. BLNK was also increased 5-fold (p=0.001) in CD27- B cells from patients with cGVHD compared to CD27- B cells from healthy individuals. Intracellular flow cytometry verified that stimulation of BCR resulted in increased expression of BLNK protein in 3 healthy individuals at 36 (p=0.01) and 60 (p=0.03) hours. To extend these finding to patients with cGVHD compared to patients without cGVHD we studied purified, un-manipulated CD19+ B cells from 10 HSCT patients who were >12 months post-transplant and off high dose steroid. cGVHD (n=4) B cells had significantly increased protein expression of BLNK (p=0.005) compared to B cells from patients without cGVHD (n=6). The protein kinase Syk, upstream of BLNK, was also significantly increased in B cells from cGVHD patients (p=0.01) compared to B cells from patients without disease. These data suggest that B cells in cGVHD are hyper-responsive to antigen (Table 1). To determine if B cells traffic to lesional tissue, liver biopsies were immunohistochemically analyzed in 3 patients with cGVHD. Two of 3 patients with disease had detectable CD27+ and CD27- B cells in the liver while 2 reference biopsies from acute hepatitis patients, without cGVHD, did not. This affirms that BCR-activated B cells can be found at cGVH disease sites. Increased expression of BLNK and Syk in B cells from patients with cGVHD may amplify subsequent BCR-signaling and contribute to the pathophysiology of cGVHD. Thus, small molecule inhibitors of Syk may represent a potential therapeutic option for patients with cGVHD.

Table 1. BLNK and Syk are increased in B cells from patients with cGVHD

Phenotype

BLNK (MFI)

Median +/- SE

p-value

Syk (MFI)

Median +/- SE

p-value

Without cGVHD

(n=6)

3.67 +/- 0.14

13.85 +/- 0.28

With cGVHD

(n=4)

4.52 +/- 0.15

0.005

16.50 +/- 1.20

0.013

cGVHD; chronic GVHD, MFI; Mean Fluorescence Intensity

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