To gain more insight about these possibilities, we repeated the experiments and examined the number of B cells proximal to the GC sites in AID

To gain more insight about these possibilities, we repeated the experiments and examined the number of B cells proximal to the GC sites in AID.Cre.ERT2.tdTomato mice in which recently GC-departed cells are labeled. context of intact lymph nodes (LNs) throughout the Dot1L-IN-1 GC response, and examined the role of BCR affinity in dictating their position. Imaging of entire GC structures and proximal single cells by light-sheet fluorescence microscopy revealed that individual B cells that previously expressed AID are located within the LN cortex, in an area close to the GC LZ. Using in situ photoactivation, we exhibited that B cells migrate from the LZ toward the GC outskirts, while DZ B cells are confined to the GC. B cells expressing very-low-affinity BCRs formed GCs but were unable to efficiently disperse within the follicles. Our findings reveal that BCR affinity regulates B cell positioning during the GC response. Introduction Generation of protective antibodies is crucial for clearance of harmful pathogens and for establishment of long-lasting immunological memory (Victora and Nussenzweig, 2012; De Silva and Klein, 2015). In response to vaccination or invading microbes, antigen-specific B cells within secondary lymphoid organs differentiate into antibody-producing cells or early memory cells or rapidly proliferate and form structures known as germinal centers (GCs; Allen et al., 2007). The main purpose of the GC response is usually to produce long-lived plasma cells (PCs) that secrete high-affinity antibodies, and memory cells that can readily elicit an efficient antibody immune response upon re-exposure to the immune stimuli (Corcoran Rabbit polyclonal to NEDD4 and Tarlinton, 2016; Weisel and Shlomchik, 2017). GCs are divided anatomically into two distinct functional zones based on the B cell density, as well as the presence of zone-specific cellular assemblies (MacLennan, 1994; Heesters et al., 2014). In the dark zone (DZ), B cells rapidly proliferate and insert mutations into their Ig variable regions followed by migration to the GC light zone (LZ), where they interact with antigen and compete for T cell help (Allen et al., 2007; Victora et al., 2010). Iterative cycles of B cells between the GC zones lead to accumulation of affinity-enhancing mutations and ultimately to progressive increase in serum antibody affinity, a process known as antibody affinity maturation (Eisen and Siskind, 1964; Jacob et al., 1991). During the GC reaction, rare B cell subsets express surface markers that identify pre-memory cells as well as transcription factors that promote the generation of Dot1L-IN-1 pre-PCs such as Irf4 and Blimp-1 (Kr?utler et al., 2017; Laidlaw et al., 2017; Suan et al., 2017a; Wang et al., 2017). PC-related markers are widely used to detect PCs positioned outside the GC structure by imaging techniques, and lineage-specific markers on non-GC class switched B cell subsets are used to detect memory cells by flow cytometry techniques (Mohr et al., 2009; Fooksman et al., 2010; Meyer-Hermann et al., 2012; Kr?utler et al., 2017; Zhang et al., 2018). It was shown that PCs are found in the proximity of the GC DZ, and Blimp-1+ or CD138+ cells were demonstrated to traverse through the T zone to the LN medullary cords at early stages of the B cell response (Fooksman et al., 2010; Meyer-Hermann et al., 2012; Zhang et al., 2018). However, the position and the path taken by GC-derived PCs at later stages of the response are less clear. In addition, it was exhibited that few memory B cells are positioned next to contracting GCs and next to the subcapsular Dot1L-IN-1 sinus, where they can rapidly respond to antigen upon re-exposure (Aiba et al., 2010; Suan et al., 2017a; Moran et al., 2018). Nonetheless, since these cells have no clear markers for imaging analysis during the GC reaction, detecting post-GC cells remains a challenge. Furthermore, cells that are not fully differentiated and do not express common memory and.