Cell sorting was performed from the University or college of Minnesota Circulation Cytometry Core using a 3-laser (407, 488, and 633 nm) FACSAria (BD)
Cell sorting was performed from the University or college of Minnesota Circulation Cytometry Core using a 3-laser (407, 488, and 633 nm) FACSAria (BD). Plasma cells do not appear to respond to a second illness because of low surface manifestation of the membrane-bound version of Ig (BCR; Manz et al., 1998). Memory space B cells, in contrast, maintain BCR manifestation and differentiate quickly into Tacalcitol monohydrate antibody-secreting cells after encountering the antigen a second time (Benson et al., 2009; Dogan et al., 2009; Pape et al., 2011). Memory space B cells are the progeny of rare naive B cells that communicate BCRs specific for the eliciting antigen. After antigen binding to the BCR and receipt of signals from helper T cells, naive B cells proliferate and undergo Ig isotype switching from IgM and IgD to IgG, IgA, or IgE (MacLennan, 1994; McHeyzer-Williams and Tacalcitol monohydrate McHeyzer-Williams, 2005; Tarlinton, 2008; Maul and Gearhart, 2010). The cells then differentiate into short-lived plasma cells that secrete antibodies, or germinal center (GC) cells, which then generate memory space B cells and long-lived plasma cells (MacLennan, 1994; McHeyzer-Williams and McHeyzer-Williams, 2005; Tarlinton, 2008; Maul and Gearhart, 2010). Memory space cells are selected in GC through a process including acquisition of Ig somatic hypermutations that enhance antigen binding and allow successful competition for survival-promoting signals from helper T cells (MacLennan, 1994; McHeyzer-Williams and McHeyzer-Williams, 2005; Tarlinton, 2008; Maul and Gearhart, 2010). Recent evidence, however, offers posed challenges to this traditional model. First, several studies possess noted the living of memory space B cells with IgM+ BCRs (Klein et al., 1997, 1998; Anderson et al., 2007; Dogan et al., 2009; Pape et al., 2011). Moreover, these IgM+ memory space cells can outnumber the isotype-switched (swIg+) memory space cells of the same specificity (Dogan et al., 2009; Pape et al., 2011). Second, Tacalcitol monohydrate memory Rabbit polyclonal to FABP3 space B cells and GC cells appear simultaneously (Blink et al., 2005; Chan et al., 2009), whereas the model predicts that GC cells should arise Tacalcitol monohydrate 1st. Lastly, not all memory space B cells have Ig somatic mutations (Schittek and Rajewsky, 1992; Anderson et al., 2007; Pape et al., 2011) and memory space B cells can be recognized in mice that cannot form GC (Toyama et al., 2002). Collectively, the data indicate that Ig isotype switching, somatic mutation, and GC selection are not required for memory space cell generation. The GC-independent pathway of memory space B cell formation, however, is not recognized. In this study, we assessed the contributions of the GC-dependent and -self-employed pathways of memory space B cell formation using an antigen-based cell enrichment protocol that we recently developed (Pape et al., 2011). We focused on very early occasions in the primary response to identify the point at which the two pathways diverged. We found that GC-independent memory space B cells were primarily CD73? and IgM+ and were derived directly from a multipotent precursor that also produced GC cells. GC cells then generated primarily swIg+ memory space B cells, which could become identified by manifestation of CD73. RESULTS Detection and phenotypic analysis of antigen-specific B cells Naive B cells specific for a given antigen are hard to detect because they are rare among the 200 106 nucleated cells in the secondary lymphoid organs of a mouse. To analyze all antigen-specific B cells in these organs by circulation cytometry, we developed a cell enrichment protocol that concentrates of the relevant cells into a sample comprising 106 cells (Pape.