Thus, effector B cell clones within a recipient mouse had been very closely related, but each recipient contained clonally distinct effector cells. Open in a separate window Figure 6. IgH Chain Repertoire Analyses of Differentiated IgM Memory CellsIgSeq analysis was performed on B cell populations generated from transferred IgM memory B cells. (A) Frequency of IgH V-gene usage in the donor IgM memory population and each IgM memory cell-derived B cell population. 2009). In our studies, few if any cells were labeled in uninfected (AID- Cre-ERT2 x Rosa26 eYFP) F1 mice, indicating that the labeled cells were contamination specific (Papillion et al., KIR2DL5B antibody 2017). Although our previous studies focused on CD11c+ IgM memory cells, eYFP+ B cells detected after tamoxifen administration were found to be more diverse. In addition to CD11c+ T-bet+ IgM memory B cells, smaller populations of differentiated GL7+ GC B cells, as well as CD138+ ASCs, were detected within the eYFP+ B cell populace (Figures ?(Figures1A,1A, top middle panel, and S1A. Nearly all of the GL7- and CD138-double-negative eYFP- labeled B cells expressed IgM (R1; i.e., are memory IgM cells), although low frequencies of swIg cells, also presumably memory L-Tyrosine cells, were detected (Physique 1A, R4). Open in a separate window Physique 1. Characterization of Aicda-Expressing IgM+ Memory Cells In VivoE.-muris-infected (AID-creERT2 ROSA26-eYFP) F1 mice were administered tamoxifen on days 7 and 10 post-infection, and splenocytes were L-Tyrosine analyzed on day 70 post-infection. (A) eYFP+ GL7neg CD138neg IgM+ memory cells (R1), CD19hi B cells (R2), CD19+ follicular B cells (R3), and eYFP+ GL7neg CD138neg IgMneg switched memory cells (R4) were recognized. Data from a representative experiment are shown in the plots at the top; the plots at the bottom are aggregate data indicating the frequency of each ofthe populations. *p 0.05, ***p 0.001, and ****p 0.0001. (B) L-Tyrosine The B cells recognized in the regions defined in (A) were monitored for their expression of a panel of markers previously characterized on IgM memory B cells (Yates et al., 2013). Cells in R1 are shown in blue and R2 in reddish; R3 cells are indicated with a black line (open histograms). (C) The expression of the indicated markers was analyzed on eYFP+ GL7neg CD138neg IgM+ memory cells (R4; orange histogram) and eYFP+ GL7neg CD138neg IgMneg memory cells (R1; blue histogram); overlapping cells appear as green. (D) The expression of CD11b was analyzed in eYFP+ GL7neg CD138neg CD11c+ (purple histogram) and CD11cneg IgM+ memory cells (green histogram). The data in (A)-(D) are representative of two experiments that used 4 mice per group. (A) Statistical significance was decided using a repeated- steps one-way ANOVA with Tukeys multiple comparison test for L-Tyrosine the left (p 0.0001; F = 0.678; df =11) and middle panels (p 0.0001; F = 0.0002; df = 11) or a two-tailed paired t test for the data in the right panel (p 0.0001;t = 59;df = 3). In (C) and (D), **p 0.01, ***p 0.001, and ****p 0.001.(E) A Venn diagram is usually shown that illustrates the relationships between the various populations that were characterized. CD11c+ and CD11cneg cells and cells expression Aicda are indicated by the colors. IgM and swIg memory cellsare indicated by cross-hatching. Observe text for details. The eYFP-labeled IgM memory cells exhibited cell surface marker expression similar to the IgM memory cells described in our previous studies (Yates et al., 2013). However, approximately 40% of the labeled IgM memory cells did not express CD11c (Physique 1B). We had not recognized these putative CD11cneg memory cells in our previous studies, which relied on the unique expression of CD11c for memory cell identification (Yates et al., 2013). Also included in the analyses were eYFPneg CD19hi B cells (Physique.