RESUMEN
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 IRF4 and IRF8, each critical to B-cell differentiation and fate. All-trans retinoic acid (ATRA) increased IRF4 expression, restored the IRF4-to-IRF8 ratio, abrogated BCR-NOTCH hyperactivation, and reduced NOTCH2 expression in cGVHD B cells without compromising viability. ATRA-treated cGVHD B cells had elevated TLR9 and PAX5, but not BLIMP1 (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.
Asunto(s)
Linfocitos B/metabolismo , Enfermedad Injerto contra Huésped/metabolismo , Trasplante de Células Madre Hematopoyéticas , Proteínas de Neoplasias/metabolismo , Receptor Notch2/metabolismo , Receptores de Antígenos de Linfocitos B/metabolismo , Transducción de Señal , Proteínas Adaptadoras Transductoras de Señales/biosíntesis , Proteínas Adaptadoras Transductoras de Señales/genética , Adulto , Anciano , Aloinjertos , Linfocitos B/patología , Enfermedad Crónica , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Enfermedad Injerto contra Huésped/genética , Enfermedad Injerto contra Huésped/patología , Neoplasias Hematológicas/genética , Neoplasias Hematológicas/metabolismo , Neoplasias Hematológicas/patología , Humanos , Factores Reguladores del Interferón/biosíntesis , Factores Reguladores del Interferón/genética , Masculino , Persona de Mediana Edad , Proteínas de Neoplasias/genética , Receptor Notch2/genética , Receptores de Antígenos de Linfocitos B/genética , Tretinoina/farmacologíaRESUMEN
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. 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.