• Human cGVHD B cells have increased proximal BCR signaling protein expression and are more BCR responsive than non-cGVHD B cells.• Inhibiting Syk kinase activity abrogates the BCR-driven ex vivo proliferative and survival advantage of human chronic GVHD B cells.Although B cells have emerged as important contributors to chronic graft-versus-hostdisease (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. 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 hyperresponsiveness 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. (Blood. 2014;123(13):2108-2115

B-cell receptor (BCR)-activated B cells contribute to pathogenesis in chronic graft-versus-host disease (cGVHD), a condition manifested by both B-cell autoreactivity and immune deficiency. We hypothesized that constitutive BCR activation precluded functional B-cell maturation in cGVHD. To address this, we examined BCR-NOTCH2 synergy because NOTCH has been shown to increase BCR responsiveness in normal mouse B cells. We conducted ex vivo activation and signaling assays of 30 primary samples from hematopoietic stem cell transplantation patients with and without cGVHD. Consistent with a molecular link between pathways, we found that BCR-NOTCH activation significantly increased the proximal BCR adapter protein BLNK. BCR-NOTCH activation also enabled persistent NOTCH2 surface expression, suggesting a positive feedback loop. Specific NOTCH2 blockade eliminated NOTCH-BCR activation and significantly altered NOTCH downstream targets and B-cell maturation/effector molecules. Examination of the molecular underpinnings of this "NOTCH2-BCR axis" in cGVHD revealed imbalanced expression of the transcription factors and, each critical to B-cell differentiation and fate. All- retinoic acid (ATRA) increased expression, restored the-to- ratio, abrogated BCR-NOTCH hyperactivation, and reduced NOTCH2 expression in cGVHD B cells without compromising viability. ATRA-treated cGVHD B cells had elevated and, but not (a gene-expression pattern associated with mature follicular B cells) and also attained increased cytosine guanine dinucleotide responsiveness. Together, we reveal a mechanistic link between NOTCH2 activation and robust BCR responses to otherwise suboptimal amounts of surrogate antigen. Our findings suggest that peripheral B cells in cGVHD patients can be pharmacologically directed from hyperactivation toward maturity.

The haematopoietic system has a highly regulated and complex structure in which cells are organized to successfully create and maintain new blood cells. It is known that feedback regulation is crucial to tightly control this system, but the specific mechanisms by which control is exerted are not completely understood. In this work, we aim to uncover the underlying mechanisms in haematopoiesis by conducting perturbation experiments, where animal subjects are exposed to an external agent in order to observe the system response and evolution. We have developed a novel Bayesian hierarchical framework for optimal design of perturbation experiments and proper analysis of the data collected. We use a deterministic model that accounts for feedback and feedforward regulation on cell division rates and self-renewal probabilities. A significant obstacle is that the experimental data are not longitudinal, rather each data point corresponds to a different animal. We overcome this difficulty by modelling the unobserved cellular levels as latent variables. We then use principles of Bayesian experimental design to optimally distribute time points at which the haematopoietic cells are quantified. We evaluate our approach using synthetic and real experimental data and show that an optimal design can lead to better estimates of model parameters.

Type 2 diabetes is the most prevalent endocrine disease in the world, and recently the gut microbiota have become a potential target for its management. Recent studies have illustrated that this disease may predispose individuals to certain microbiome compositions, and treatments like metformin have been shown to change gut microbiota and their associated metabolic pathways. However, given the limitations and side effects associated with pharmaceuticals currently being used for therapy of diabetes, there is a significant need for alternative treatments. In this study, we investigated the effects of a root extract from Rhodiola rosea in a Leptin receptor knockout (db/db) mouse model of type 2 diabetes. Our previous work showed that Rhodiola rosea had anti-inflammatory and gut microbiome-modulating properties, while extending lifespan in several animal models. In this study, treatment with Rhodiola rosea improved fasting blood glucose levels, altered the response to exogenous insulin, and decreased circulating lipopolysaccharide and hepatic C-reactive protein transcript levels. We hypothesize that these changes may in part reflect the modulation of the microbiota, resulting in improved gut barrier integrity and decreasing the translocation of inflammatory biomolecules into the bloodstream. These findings indicate that Rhodiola rosea is an attractive candidate for further research in the management of type 2 diabetes.

Patients with chronic graft versus host disease (cGVHD) have persistently altered B cell homeostasis and loss of B cell tolerance, even years after allogeneic hematopoietic stem cell transplantation (HSCT). Chronic GVHD patients have been shown to have a diverse group of autoantibodies. In cGVHD patients, antigen-experienced CD27+ B cells, unlike CD27- B cells, are capable of constitutive IgG secretion without the need for ex vivo stimulation [Sarantopoulos et al., Blood. 2009]. We previously showed that potentially allo- or auto-reactive B cells in human cGVHD signal via BAFF-associated pathways [Allen et al., Blood. 2012]. B cell receptor (BCR) repertoire composition in cGVHD B cell subsets remains unknown. We hypothesized that potentially pathologic CD27+ B cell subsets could be identified in cGVHD patients by sequencing of the immunoglobulin heavy chain (IGH). In our study, high-throughput sequencing of the IGH was performed after preparation of single molecule real time (SMRT) B-cell amplicon libraries from RT-PCR products per Pacific Biosciences 500bp protocol, using framework region 2 (FR2) IGH variable (V) gene family primers and a common IGH Joining (J) gene primer [Boyd et al., Science Translational Medicine. 2009]. Sufficient numbers of purified B cell subsets, only available from large volume cGVHD leukapheresis samples, were obtained from three cGVHD patients and three healthy donors (HDs). In each cGVHD patient and in HDs, we examined the following CD27+ and CD27- B cell populations: naïve/transitional (CD27- IgD+ CD38+), double negative (CD27- IgD- CD38+), CD27+ IgD+ memory (CD27+ IgD+ CD38+) and CD27+ IgD- memory (CD27+ IgD- CD38+). The IGH complementarity-determining region 3 (CDR3) is crucial for BCR antigen-specificity, and CDR3 characteristics have been previously shown to associate with autoreactivity [Wardemann et al., Science. 2003]. Thus, we assessed the CDR3 characteristics (length, charge, amino acid composition, hydrophobicity) in each B cell subset in HDs and cGVHD patients. We found that cGVHD CD27+ IgD+ B cells had overall CDR3 amino acid charge and length similar to naïve/transitional B cells. Since the CDR3 sequence is a result of V and J segment joining and may associate with capacity for autoreactivity, we also assessed relative V and J gene family usage. Gene usage analyses showed that the IGHV3 gene contributes to the majority of IGHV in all subsets of both cGVHD patients and HD groups, validating a previous report of three healthy individuals [Wu et al., Blood. 2010]. Notably, we found a 10-fold increase in frequency of IGHV7 usage in cGVHD patients compared to HD B cell subsets (1.13% vs. 0.09 in naïve/ transitional, 1.1% vs. 0.06 in double negative, 1.05 vs. 0.8 in CD27+ IgD+ memory and 0.8 vs. 0.06 in CD27+ IgD- memory). Additionally, the previously well-described autoreactive gene IGHV4-34 was more frequent in cGVHD patients compared to HD in all B cell subsets except in double negative cells. Interestingly, IGHV4-34 usage was particularly frequent in the CD27+ IgD+ cells, with a mean value of 33.1% compared to 23.3% in healthy donors. Consistent with autoreactivity, plasma IgG from these cGVHD patients had positive HEp-2 cell staining. Of the four B cell subsets examined, the CD27+ IgD+ memory in cGVHD had other distinct IGH characteristics. Additionally, IgD+ CD27+ memory B cells from cGVHD patients displayed a relative increase in IGHJ6 usage, with 37.1% compared to the 26.7% found in HD. Higher usage of tyrosine in CD27+ IgD+ population corroborated this finding. Taken together, the IgD+ CD27+ B cell subset possessed an IGH repertoire with three features unique to cGVHD: 1) similarity to the naïve/transitional cells in the CDR3 length and total charge, 2) increased IGHJ6 usage, and 3) increased IGHV4-34 gene usage. We previously demonstrated increased cell size in CD27+ B cells, in particular in the pre-Germinal Center (GC) subset that was included in the IgD+ CD27+ gate in the current analysis. Of note, pre-GC cells were previously found to uniquely circulate in cGVHD and express high levels of the BAFF receptors TACI and BCMA [Sarantopoulos et al., Blood. 2009], corroborating the potential autoreactive capacity of this cell population. Thus, our current data suggest that IgD+CD27+ B cells in cGVHD patients display distinctly autoreactive features, and are a potentially pathologic B-cell subset in cGVHD. Disclosures: No relevant conflicts of interest to declare.