Rhinovirus-Specific B-Cells Display Signature and Functionality Consistent With Age-Associated B-Cells

Abstract

RATIONALE: Human rhinoviruses (RV) account for half a billion colds annually in the US, and exacerbate chronic respiratory diseases. Repeated infections continue into adulthood, owing to the lack of a durable protective antibody response that remains ill-understood. Here, we sought to rigorously define B-cell responses to RV using novel serologic and flow cytometric immunoassays. METHODS: Serum antibodies were assayed in 16 subjects before (day 0) and after (day 21) experimental infection with RV-A16 using bead-bound capsid proteins and purified whole virus. RV-specific B-cells were isolated from 6 subjects with a history of natural RV infection by labeling with fluorescent virus. Their capacity to secrete antibodies was assessed in vitro by stimulating FACS-sorted cells with soluble anti-CD40 and CpG-DNA, and analyzing supernatants on days 2, 4, 6, and 8. RESULTS: After infection, significant increases in multiple serum antibody isotypes were restricted to whole virus (IgG: p<0.01; IgA: p<0.01; and IgM: p<0.05), and all isotypes cross-reacted with a second RV-A strain. Virus-labeled B-cells were enriched for ''Age-Associated Bcells'' (ABC, ;20%) based on expression of T-bet, CD11c, CD21, CXCR5, and IgG. Additionally, 5.0+/-2.7% of total ABCs were RVspecific, and RV-specific ABCs bound multiple RV-A strains. The capacity of RV-specific ABCs to secrete antibodies was reduced compared with other RV-specific memory cells. CONCLUSIONS: RV infection induces cross-reactive antibodies directed against intact capsid, and disproportionately expands hypofunctional ABCs. Given that ABCs may be exhausted in chronic viral infections, their expansion suggests the induction of anergy in broadlyreactive B-cells, as a consequence of repeated RV infections. RATIONALE: Chronically inflamed airways in asthma and chronic obstructive pulmonary disease (COPD) are susceptible to heightened neutrophilic inflammation but the underlying mechanisms are unclear. We previously showed that SP-D deficiency is associated with increased and prolonged neutrophilic inflammation of the airways in mice. Extracellular release of double stranded DNA by neutrophils contributes to the inflammatory injury. We hypothesized that SP-D interferes with this process. METHODS: To study the role of SP-D in airway neutrophilia we exposed wild-type and SP-D-/-mice to ozone (3 ppm, 2 hours) and studied the cellular and molecular inflammatory changes. Bronchoalveolar lavage (BAL) SP-D was investigated by the biotin switch assay and Western blot. Mouse and human neutrophils were studied in vitro for SP-D binding and DNA release by immunochemistry and PicoGreen fluorescence quantitation. RESULTS: SP-D-/-mice had increased myeloperoxidase gene and protein expression in the lung suggesting presence of activated neutrophils. In the BAL of O 3 exposed mice neutrophilic airway inflammation was associated with de-oligomerisation and cysteine nitrosylation of the SP-D molecule. Using recombinant SP-D and confocal imaging, we found that SP-D directly bound to the membrane and ...