Fig 2: Early cell cycle exit and induction of MBC marker expression on antigen-specific B cells in immunized rhesus macaquesRhesus macaques were immunized with AF647 fluorophore-labeled RSVF immunogens (provided as nanoparticles or soluble vaccine) by intramuscular injection to study the vaccine-draining LNs 2 h, 3 days or 7 days after immunization (STAR Methods).(A) Flow cytometric analysis of LNs 7 days after nanoparticle immunization. B-lineage cells were gated asCD20+ and/or IRF4+, and antigen-specific B cells were identified by dual staining with RSVF-AF488 and RSVF-BV421 tetramer probes. Intracellular expression of BCL6, IRF4, Ki67, IgM, and IgG as well as surface expression of CCR6, CD27, and CD95 by antigen-specific B cell subpopulations (gated as indicated) are shown. Quantification is shown for individual draining LNs of the same immunized animal. Downregulation of Ki67 expression by antigen-specific B cells was confirmed in cryo-preserved samples from two additional animals (data not shown) at day 7 after immunization. Horizontal lines indicate the mean, and error bars represent SD.(B) Representative images of the fluorescent soluble vaccine (red) localization in LNs stained for CD3 expression (white) to visualize LN structure. LNs after 2 h, 3 days, and 7 days after immunization are shown (left and center). Shown is a representative image of a LN follicle displaying co-localization of the vaccine (red) with CD35+ follicular dendritic cells (cyan) (right).See also Figure S7.

Fig 3: Early cell cycle exit of eMBCs takes place with physiologically low numbers of naive B cells(A) OVA-primed WT mice were transferred with congenically distinguishable B1-8hi splenocytes containing an estimated number of 5 × 105, 5 × 104, or 5 × 103 NP-specific B cells. The population composition of Ig?+ B1-8hi cells and Ki67 expression in the CCR6+ population was analyzed at day 4 after i.p. immunization with NP-OVA in alum. Shown are representative results of two independent experiments.(B) WT mice were immunized i.p. with NP-OVA in alum. NP-specific cells among Ig?+ B cells were detected by dual staining with NP-PE and NP-BSA-bio/Strep-BV650. Intracellular staining for BCL6, IRF4, and Ki67 was performed, and Ki67 expression in GCBC, PB, and AP-eMBC populations was analyzed as indicated. **p < 0.01 (paired one-way ANOVA with Geisser-Greenhouse correction). Horizontal lines indicate the mean and error bars represent SD.(C) WT or S1pr2-ERT2cre R26tdTomato/+ mice were immunized i.p. with NP-OVA in alum, and S1pr2-ERT2cre R26tdTomato/+ mice were treated by oral gavage with tamoxifen at days 2, 4, and 6 after immunization. Expression of tdTomato at day 7 is shown for the NP-specific (NP-BSA-bio/Strep-BV650+) Ig?+ PB (TACI+-CD138+), GCBC (GL7hiIg?int), and AP-eMBC (GL7lo/intIg?hi) populations. Shown are representative results of two independent experiments.(D) Mice were treated as in (C) but for up to 45 days (tamoxifen treatment every 2 days for the duration of the experiment). NP-specific cells among Ig?+ B cells were detected with dual staining using NP-CGG-AF488 and NP-BSA-bio/Strep-PECy7 (STAR Methods). Frequencies of tdTomato+ cells among MBCs and GCBCs at the indicated time points are shown. Shown are representative results of two independent experiments. Horizontal lines indicate the mean, and error bars represent SD.(E) Mice were infected intranasally with IAV. Cells from mediastinal LNs pooled from nine animals at day 7 post infection were analyzed by flow cytometry. Binding of hemagglutinin (HA) probes to B-lineage cells (CD19+ and/or CD138+) and intracellular expression of IRF4, BCL6, and Ki67 in HA-specific B cells is shown. Splenic naive B cells (CD19+IgD+) of a non-infected mouse were used as a control. Shown are representative results of two independent experiments.See also Figure S6.