Panel A: Flow cytometry plots show the gating strategy for purifying marginal zone (MZ) B cells from spleens of WT:WT (wild-type to wild-type) and KO:WT (knockout to wild-type) chimeras. The plots display markers including CD19 (B cell marker), CD93 (immature B cell marker), CD23 (low-affinity IgE receptor), and CD45.1 (leukocyte common antigen isoform). Panel B: Flow cytometry plots illustrate the gating strategy to identify CD45.2 positive and CD45.1 positive plasmablasts (antibody-secreting precursor cells) after 3 days of in vitro LPS (lipopolysaccharide) activation of MZ B cells, using markers such as CD138 (plasma cell marker) and B220 (B cell marker). Panel C: Flow cytometry plots depict the gating strategy to identify NP-specific (nitrophenyl antigen-specific) CD45.2 positive and CD45.1 positive splenic germinal center B cells (GCB) 7 days post-immunization with NP-CGG (nitrophenyl–chicken gamma globulin), using markers such as CD19 (B cell marker), CD138 (plasma cell marker), FAS (CD95, apoptosis receptor), GL7 (germinal center marker), IgD (Immunoglobulin D), and CD38 (activation marker). Panel D: A bar graph shows the numbers of NP-specific CD45.2 positive and CD45.1 positive splenic germinal center B cells in WT:WT and KO:WT chimeras 7 days post-immunization. The x-axis represents conditions (phosphate-buffered saline (PBS) or NP-CGG), and the y-axis represents cell numbers. Panel E: Flow cytometry plots show the gating strategy to identify EdU positive (5-ethynyl-2?-deoxyuridine labeled proliferating cells) CD45.2 positive follicular (FO) and marginal zone (MZ) B cells in splenocytes from WT:WT and KO:WT chimeras after continuous in vivo EdU labeling. Panel F: Line graphs display the proportion of EdU positive cells within T1 (transitional 1), T2 (transitional 2), and T3 (transitional 3) CD45.2 positive immature splenic B cell populations at different time points (4 hours, 2 days, 3 days, 5 days). The x-axis represents time, and the y-axis represents the percentage of EdU positive cells. Panel G: Flow cytometry plots illustrate the gating strategy to identify transferred CD45.2 positive and CD45.1 positive follicular (FO) and marginal zone (MZ) B cells in the spleens of recipient mice 7 days after adoptive transfer. Panel H: Flow cytometry plots show the gating strategy to identify transferred CMFDA-labeled (CellTracker Green 5-chloromethylfluorescein diacetate stained) CD45.2 positive and CD45.1 positive follicular (FO) and marginal zone (MZ) B cells in the spleens of recipients 1 hour after adoptive transfer.
Analysis of plasmablast differentiation, NP-CGG immunization, EdU labeling, and adoptive transfer studies. (A) Gating strategy used to purify MZ B cells from the spleens of WT:WT and KO:WT chimeras by FACS for in vitro plasmablast differentiation assays. (B) Gating strategy used to identify CD45.2+ and CD45.1+ plasmablasts (CD138+B220lo) after 3 days of in vitro LPS activation of MZ B cells. (C) Gating strategy used to identify NP-specific CD45.2+ and CD45.1+ splenic GCBs (CD19+CD138−FAS+GL7hiIgD−CD38loNP+) 7 days after immunization of WT:WT and KO:WT chimeras with PBS or NP-CGG with alum. (D) Numbers of NP-specific CD45.2+ and CD45.1+ splenic GCBs in WT:WT and KO:WT chimeras, 7 days after immunization with PBS or NP-CGG with alum. Bars show the mean of n = 8 (WT:WT +PBS), n = 7 (KO:WT +PBS), or n = 10 (WT:WT and KO:WT +NP-CGG) mice, pooled from two independent experiments. Each point represents one mouse. Black: WT CD45.2+; red: TACI KO CD45.2+; gray: WT CD45.1+. Statistical test: two-way ANOVA with Šídák’s multiple comparisons test. Numbers above graphs indicate P values where P ≤ 0.05, otherwise no value is shown. (E) Gating strategy used to identify EdU+ CD45.2+ FO and MZ B cells in splenocytes from WT:WT and KO:WT chimeras after continuous EdU labeling in vivo. (F) Proportion of EdU+ cells within T1 (IgDlo), T2 (IgD+IgM+), and T3 (IgDhiIgMlo) CD45.2+ immature splenic B cell populations (CD93+B220+CD19+) from WT:WT and KO:WT chimeras at 4 h, and 2, 3, and 5 days after EdU injection (performed at 12 wk after reconstitution) and EdU continuously administered via drinking water. Each point represents one mouse (4 h WT, n = 4; KO n = 5; 2, 3, and 5 days, n = 4). Lines connect the mean at each time point. Black: WT CD45.2+; red: TACI KO CD45.2+. No difference reported between WT and KO means at any time point as determined by two-way ANOVA with Šídák’s multiple comparisons test. (G) Gating strategy used to identify transferred CD45.2+ and CD45.1+ FO and MZ B cells in the spleens of CD45.2+CD45.1+ recipients, 7 days after adoptive transfer. (H) Gating strategy used to identify transferred CMFDA-labeled CD45.2+ and CD45.1+ FO and MZ B cells in the spleens of CD45.2+CD45.1+ recipients, 1 h after adoptive transfer. GCBs, germinal center B cells.