SummaryOne manifestation of fluorescence resonance energy transfer (FRET) is an increase in donor fluorescence after photobleaching the acceptor. Published acceptor-photobleaching methods for FRET have mainly used wide-field microscopy. A laser scanning confocal microscope enables faster and targeted bleaching within the field of view, thereby improving speed and accuracy. Here we demonstrate the approach with CFP and YFP, the most versatile fluorescent markers now available for FRET. CFP/YFP FRET imaging has been accomplished with a single laser (argon) available on virtually all laser-scanning confocal microscopes. Accordingly, we also describe the conditions that we developed for dual imaging of CFP and YFP with the 458 and 514 argon lines. We detect FRET in a CFP/YFP fusion and also between signalling molecules (TNF-ReceptorAssociated-Factors or TRAFs) that are known to homo-and heterotrimerize. Importantly, we demonstrate that appropriate controls are essential to avoid false positives in FRET by acceptor photobleaching. We use two types of negative control: (a) an internal negative control (non-bleached areas of the cell) and (b) cells with donor in the absence of the acceptor (CFP only). We find that both types of negative control can yield false FRET. Given this false FRET background, we describe a method for distinguishing true positive signals. In summary, we extensively characterize a simple approach to FRET that should be adaptable to most laser-scanning confocal microscopes, and demonstrate its feasibility for detecting FRET between several CFP/YFP partners.

Both constitutive Ig secretion by long-lived plasma cells (PC) and the recurrent differentiation of memory (mem) B cells into PC contribute to the maintenance of serologic mem. However, the relative contribution of each is unknown. In this study, we describe a novel population of human postswitched mem B cells that rapidly differentiate into PC and thus contribute to serologic mem. These IgG+ B cells reside in the region of human spleen analogous to the murine marginal zone and have not previously been examined. These cells are highly responsive to IL-21 in the context of CD40 stimulation. Uniquely, IgG+ marginal zone analog B cells are exquisitely sensitive to the combination of IL-21 and B cell-activating factor belonging to the TNF family (BAFF/BLyS) that synergize in the absence of further costimulation to induce up-regulation of B lymphocyte-induced maturation protein-1 and drive PC differentiation. Other cytokine combinations are not active in this regard. This is the first demonstration that this unique population of mem B cells can respond specifically and exclusively to IL-21 and BAFF/BLyS by differentiating into IgG-secreting PC, and thus contributing to serologic mem in an Ag-independent manner.

Interferon (IFN) consensus sequence-binding protein/IFN regulatory factor 8 (IRF8) is a transcription factor that regulates the differentiation and function of macrophages, granulocytes, and dendritic cells through activation or repression of target genes. Although IRF8 is also expressed in lymphocytes, its roles in B cell and T cell maturation or function are ill defined, and few transcriptional targets are known. Gene expression profiling of human tonsillar B cells and mouse B cell lymphomas showed that IRF8 transcripts were expressed at highest levels in centroblasts, either from secondary lymphoid tissue or transformed cells. In addition, staining for IRF8 was most intense in tonsillar germinal center (GC) dark-zone centroblasts. To discover B cell genes regulated by IRF8, we transfected purified primary tonsillar B cells with enhanced green fluorescent protein–tagged IRF8, generated small interfering RNA knockdowns of IRF8 expression in a mouse B cell lymphoma cell line, and examined the effects of a null mutation of IRF8 on B cells. Each approach identified activation-induced cytidine deaminase (AICDA) and BCL6 as targets of transcriptional activation. Chromatin immunoprecipitation studies demonstrated in vivo occupancy of 5′ sequences of both genes by IRF8 protein. These results suggest previously unappreciated roles for IRF8 in the transcriptional regulation of B cell GC reactions that include direct regulation of AICDA and BCL6.