Heightened TLR7 signaling primes BCR-activated B cells in chronic graft-versus-host disease for effector functions.

ABSTRACT: Chronic graft-versus-host disease (cGVHD) is a debilitating, autoimmune-like syndrome that can occur after allogeneic hematopoietic stem cell transplantation. Constitutively activated B cells contribute to ongoing alloreactivity and autoreactivity in patients with cGVHD. Excessive tissue damage that occurs after transplantation exposes B cells to nucleic acids in the extracellular environment. Recognition of endogenous nucleic acids within B cells can promote pathogenic B-cell activation. Therefore, we hypothesized that cGVHD B cells aberrantly signal through RNA and DNA sensors such as Toll-like receptor 7 (TLR7) and TLR9. We found that B cells from patients and mice with cGVHD had higher expression of TLR7 than non-cGVHD B cells. Using ex vivo assays, we found that B cells from patients with cGVHD also demonstrated increased interleukin-6 production after TLR7 stimulation with R848. Low-dose B-cell receptor (BCR) stimulation augmented B-cell responses to TLR7 activation. TLR7 hyperresponsiveness in cGVHD B cells correlated with increased expression and activation of the downstream transcription factor interferon regulatory factor 5. Because RNA-containing immune complexes can activate B cells through TLR7, we used a protein microarray to identify RNA-containing antigen targets of potential pathological relevance in cGVHD. We found that many of the unique targets of active cGVHD immunoglobulin G (IgG) were nucleic acid-binding proteins. This unbiased assay identified the autoantigen and known cGVHD target Ro-52, and we found that RNA was required for IgG binding to Ro-52. Herein, we find that BCR-activated B cells have aberrant TLR7 signaling responses that promote potential effector responses in cGVHD.

Enhancing Adolescent Reproductive Health Services: Assessment of Health Care Practices in Holyoke and Springfield, MA Engaged in the Youth First Initiative.

Health care practices can play a key role in reducing teen pregnancies, though current health care systems do not adequately meet adolescents' reproductive health needs. To address this gap, Youth First, a Centers for Disease Control and Prevention funded, community-wide initiative in Holyoke and Springfield (Massachusetts) established partnerships with nine local health care practices to increase adolescent access to health services. However, we had limited knowledge about their reproductive health services and policies. To address this gap, assessments were conducted with staff using structured interviews and surveys to inform targeted efforts to enhance the quality and youth friendliness of adolescent reproductive health services. Findings revealed that many of the youth-friendly services best practices recommended by the CDC were not routinely implemented by all health care practices. Findings from this assessment can be used to support health care practices to facilitate widespread adoption of best practices related to meeting adolescents' reproductive health needs.

B cells from patients with chronic GVHD are activated and primed for survival via BAFF-mediated pathways.

Recent data reveal an important role for B cells in the pathogenesis of chronic GVHD (cGVHD). Patients with cGVHD have delayed B-cell reconstitution and elevated BAFF to B-cell ratios compared to patients without cGVHD. The mechanisms promoting and sustaining B-cell activation in this disease, however, remain unknown. As BAFF increases murine B-cell metabolism and survival and maintains autoreactive B-cell clones, we performed ex vivo analyses of peripheral B cells from 51 patients who either had or did not have active cGVHD and were greater than 1 year from the time of allogeneic hematopoietic stem cell transplantation. We found that B cells from patients with active cGVHD were in a heightened metabolic state and were resistant to apoptosis. Exogenous BAFF treatment amplified cell size and survival in B cells from these patients. We found significantly increased signaling through ERK and AKT that associated with decreased levels of proapoptotic Bim, suggesting a mechanistic link between elevated BAFF levels and aberrant B-cell survival. Thus, we identify a role for BAFF in the pathogenesis of cGVHD and define B-cell activation and survival pathways suitable for novel therapeutic development in cGVHD.

Recovery of B-cell homeostasis after rituximab in chronic graft-versus-host disease.

Investigation of the effects of rituximab (anti-CD20) on B-cell-activating factor of the tumor necrosis factor family (BAFF) and B cells would better define the significance of B-cell homeostasis in chronic graft-versus-host disease (cGVHD) pathophysiology. We studied 20 cGVHD patients at a median of 25 months after rituximab treatment when most patients had recovered total B-cell numbers. A total of 55% of patients had stable/improved cGVHD, and total B-cell numbers in these patients were significantly higher compared with rituximab-unresponsive patients. Although total B-cell number did not differ significantly between cGVHD groups before rituximab, there was a proportional increase in B-cell precursors in patients who later had stable/improved cGVHD. After rituximab, BAFF levels increased in all patients. Coincident with B-cell recovery in the stable/improved group, BAFF/B-cell ratios and CD27(+) B-cell frequencies decreased significantly. The peripheral B-cell pool in stable/improved cGVHD patients was largely composed of naive IgD(+) B cells. By contrast, rituximab-unresponsive cGVHD patients had persistent elevation of BAFF and a predominance of circulating B cells possessing an activated BAFF-R(Lo)CD20(Lo) cell surface phenotype. Thus, naive B-cell reconstitution and decreased BAFF/B-cell ratios were associated with clinical response after rituximab in cGVHD. Our findings begin to delineate B-cell homeostatic mechanisms important for human immune tolerance.

Altered B-cell homeostasis and excess BAFF in human chronic graft-versus-host disease.

Chronic graft-versus-host disease (cGVHD) causes significant morbidity and mortality in patients otherwise cured of malignancy after hematopoietic stem cell transplantation (HSCT). The presence of alloantibodies and high plasma B cell-activating factor (BAFF) levels in patients with cGVHD suggest that B cells play a role in disease pathogenesis. We performed detailed phenotypic and functional analyses of peripheral B cells in 82 patients after HSCT. Patients with cGVHD had significantly higher BAFF/B-cell ratios compared with patients without cGVHD or healthy donors. In cGVHD, increasing BAFF concentrations correlated with increased numbers of circulating pre-germinal center (GC) B cells and post-GC "plasmablast-like" cells, suggesting in vivo BAFF dependence of these 2 CD27(+) B-cell subsets. Circulating CD27(+) B cells in cGVHD comprised in vivo activated B cells capable of IgG production without requiring additional antigen stimulation. Serial studies revealed that patients who subsequently developed cGVHD had delayed reconstitution of naive B cells despite persistent BAFF elevation as well as proportional increase in CD27(+) B cells in the first year after HSCT. These studies delineate specific abnormalities of B-cell homeostasis in patients with cGVHD and suggest that BAFF targeting agents may be useful in this disease.

High levels of B-cell activating factor in patients with active chronic graft-versus-host disease.

PURPOSE: Recent studies suggest that donor B cells as well as T cells contribute to immune pathology in patients with chronic graft-versus-host disease (GVHD). B-cell activating factor (BAFF) promotes survival and differentiation of activated B cells. Thus, we tested whether BAFF correlated with chronic GVHD disease activity and time of onset after allogeneic hematopoietic stem cell transplantation (HSCT). EXPERIMENTAL DESIGN: Patients who had undergone allogeneic HSCT between 1994 and 2005 for hematologic malignancies were studied. ELISA was used to measure plasma BAFF levels and flow cytometry was used to assess BAFF receptor expression on B cells in patients with or without chronic GVHD. RESULTS: In 104 patients, BAFF levels were significantly higher in patients with active chronic GVHD compared with those without disease (P = 0.02 and 0.0004, respectively). Treatment with high-dose prednisone (>or=30 mg/d) was associated with reduced BAFF levels in patients with active chronic GVHD (P = 0.0005). Serial studies in 24 patients showed that BAFF levels were high in the first 3 months after HSCT but subsequently decreased in 13 patients who never developed chronic GVHD. In contrast, BAFF levels remained elevated in 11 patients who developed chronic GVHD. Six-month BAFF levels >or=10 ng/mL were strongly associated with subsequent development of chronic GVHD (P < 0.0001). Following transplant, plasma BAFF levels correlated inversely with BAFF receptor expression on B cells (P = 0.01), suggesting that soluble BAFF affected B cells through this receptor. CONCLUSION: These results suggest that elevated BAFF levels contribute to B-cell activation in patients with active chronic GVHD.