ABSTRACT
We previously showed that IgH sequence alone minimally influenced germinal centre (GC) B-cell survival fate. As end-stage effector B cells are typically more mutated than founder GC B cells, we worked to develop an assay that would enrich for populations of GC B cells with progressively increasing numbers of somatic mutations, which could potentially be used as an indicator of positive selection. We targeted CD45 as it has been shown to influence activation-induced cytidine deaminase (AID) expression. In this study, anti-CD77 and anti-CD45RO (RO) were used to subdivide CD19(+)IgD(-)CD38(+)CD77(+) centroblasts (CB) and CD19(+)IgD(-)CD38(+)CD77(-) centrocytes (CC) into three contiguous RO fractions (RO(-), RO(+/-) and RO(+)) and assessed whether mutation frequency and characteristics associated with selection varied with respect to increasing RO expression. Here, we show that the average number of mutations per IgV(H)4 transcript increased concordantly with RO for CC, but not for CB. CC also exhibited an RO-associated increase in replacement mutations. Comparative analysis of clonally related sequences revealed that increased mutations were not due to the exclusive persistence of surface RO on highly mutated cells. RO-expressing CC and CB pools showed increased signs of activation (CD69(+)) and were enriched for surface Ig(+) cells. BCR-crosslinking induced a significant increase in surface RO on total tonsillar and GC B cells, which collectively suggests that the RO-associated increase in mutations is attributable, at least in part, to the cycling of cells that may have recently undergone BCR-mediated selection, or are potentially in developmental transition between CC and CB stages.
Subject(s)
B-Lymphocyte Subsets/immunology , B-Lymphocyte Subsets/metabolism , Clonal Deletion/immunology , Leukocyte Common Antigens/metabolism , Receptors, Antigen, B-Cell/physiology , B-Lymphocyte Subsets/cytology , Biomarkers/metabolism , Cell Cycle/genetics , Cell Cycle/immunology , Cell Differentiation/genetics , Cell Differentiation/immunology , Child , Child, Preschool , Clonal Deletion/genetics , Gene Rearrangement, B-Lymphocyte, Heavy Chain , Germinal Center/cytology , Germinal Center/immunology , Germinal Center/metabolism , Humans , Infant , Receptors, Antigen, B-Cell/genetics , Somatic Hypermutation, ImmunoglobulinABSTRACT
Traditional CQI tools have proven effective in improving discrete processes, but the strengths of these tools often render them inappropriate to improve complex systems made up of multiple processes. After finding that CQI tools failed to reduce emergency department waiting times, a QI team applied systems thinking tools and learned that the cause of delays was rooted in interrelationships among processes, such as the impact on laboratory testing when emergent and routine patients arrived back to back. Computer modeling allowed the team to reject some quick fixes--such as adding ED beds--that simulations showed would be ineffective. By reorganizing patient flow and automating hospitalwide bed control, the team so far has reduced waiting times 19 percent and increased patient satisfaction despite an increase in census.