Fig 2: MSR vaccine generates potent humoral and cellular immune responses against a model antigen, OVA(a,b) ELISA analysis of sera OVA-specific IgG2a (a) or IgG1 (b) after immunization with bolus OVA, soluble components of the vaccine (bolus vaccine), MSRs loaded with OVA, or MSR vaccine, respectively (n=5). (c,d) ELISA analysis of sera OVA-specific (c) IgG2a and (d) IgG1 after immunization with bolus OVA, bolus vaccine formulation (GM-CSF, CpG, OVA), Imject Alum with OVA, MSR with OVA, and the MSR vaccine formulation on day 0, and boosted with the same formulations for each condition on day 30 (n=5). In (a–d), comparisons were made between vaccine and bolus formulation groups. (e,f) Flow cytometric analysis of proliferation of OVA-specific CD4+ T cells (gated on Thy1.2+ CD3+ T cells) (e) and CD4+ CXCR5+ T follicular helper (TFH) cells (gated on Thy1.2+ CD3+ T cells) (f) in naïve mice, or mice at day 3 post immunization with MSR loaded with non-specific antigen (MSR + Lysozyme), MSR loaded with OVA (MSR + OVA), or complete MSR vaccine (Vaccine) (n=4). (g,h) Number of tetramer+ CD8+ T cells (g) and IFN-?+ CD8+ T cells (h) in spleen 7 days after vaccination with blank MSR (Blank) or complete MSR vaccine (Vaccine) (n=4). (i) Flow cytometric analysis of proliferation of OVA-specific CD8+ T cells in the draining lymph node (dLN; left) and spleen (right) of naïve mice, and mice at day 3 post immunization with complete MSR vaccine (Vaccine). (j,k) Prophylactic cancer vaccine study using injectable MSRs. (j) EG.7-OVA tumor volume and (k) survival rate after subcutaneous injection of various vaccine formulations 10 days before tumor inoculation (n=10). In (j), the tumor volumes were compared on day 21, 23 and 25, following the onset of tumor growth in the vaccine group. Biological replicates were used and studies were repeated at least 2 times in the lab. Values represent mean and S.D. with exception of Fig. 5j, which is expressed as mean ± SEM. *, **, and *** represents p<0.05, p<0.01, and p<0.001, respectively.

Fig 3: Early lineage split of PBs takes place in a transient wave(A) scRNA-seq was performed as in Figure 1 but with B1-8hi cells sorted at day 2.5 after immunization. The assignment of cells to different cell types (left) and phases of the cell cycle (right) is shown on a UMAP plot.(B) Expression of class-switch recombination-related genes is shown on a UMAP plot as in (A).(C) Expression of IgM and IgG1 on Ig?+ B1-8hi APs (CCR6+) and PBs (IRF4+CD138+) at day 2.5 after immunization. Experimental setup as in Figure 1A. Pooled data from three independent experiments. *p < 0.05 (paired two-tailed Student’s t test).(D–F) Analysis of the combined scRNA-seq data of day 2.5 and 3.5–4.(D) UMAP plot showing the contribution of cells from the different time points and expression of the indicated marker genes.(E) Trajectory analysis with the Slingshot package (STAR Methods). Trajectories are plotted on a UMAP plot (left), smoothed expression of the marker genes in cells sorted by pseudotime along the indicated trajectories is shown (center and right).(F) RNA velocity analysis of the combined day 2.5/3.5–4 dataset was performed and visualized as in Figure 1F.(G) Unprimed WT mice were transferred with congenically distinguishable B1-8hi splenocytes, immunized i.p. with NP-OVA in alum, and analyzed at day 2.5 and 5 after immunization. Top: expression of CD138 and IRF4 by Ig?+ B1-8hi B cells was used to identify PBs. Bottom: expression of CD22 and MHC class II on day 2.5 PBs, day 5 PBs and non-PB Ig?+ B1-8hi cells (day 5). Shown are representative results of two independent experiments.See also Figure S3.

Fig 4: Early cell cycle exit defines the separation of the eMBC lineage(A–D) OVA-primed WT mice were transferred with congenically distinguishable CTV-labeled B1-8hi splenocytes and immunized i.p. with NP-OVA in alum. Mice were injected with EdU 14 and 4 h before analysis at the indicated time points.(A) Experimental design.(B) CTV dilution by Ig?+ and Ig?- B1-8hi B cells.(C) EdU incorporation by CCR6+ (AP-eMBC) or CCR6-GL7+ (GCBC) Ig?+ B1-8hi B cells at the indicated time points. Shown are representative contour plots (left) and quantification (right).(D) Ki67 expression by cells gated as in (C). Shown are representative histograms indicating gating of Ki67lo cells (left), frequencies of Ki67lo cells (center), and Ki67 median fluorescence intensity (MFI) (right).(B–D) Representative results of two independent experiments (B and D) and one experiment (C). **p < 0.01, ***p < 0.001, ****p < 0.0001 (one-way ANOVA with Holm-Sidak’s multiple comparisons test). Horizontal lines indicate the mean and error bars represent SD.(E) UMAP plots as in Figures 1C and 1D, highlighting expression of MBC-related genes.(F) Dot plots showing expression of MBC signature genes (top) (Laidlaw et al., 2017) and MBC transcription factor genes (bottom) (He et al., 2017) in the AP-eMBC, GCBC, and PB populations of the scRNA-seq data (day 3.5–4; Figure 1C).(G) OVA-primed WT mice were transferred with B1-8hi cells, and EdU was injected on days 1, 2, and 3 after immunization. EdU incorporation was analyzed 4 days, 4 weeks, and 8 weeks after immunization. Expression of IgM and IgG1 by EdU+ cells is shown. Horizontal lines indicate the mean and error bars represent SD.(H) OVA-primed WT mice were transferred with S1pr2-ERT2Cre R26tdTomato/+ B1-8hi cells, immunized i.p. with NP-OVA in alum, and treated with tamoxifen from day 2 every two days for the duration of the experiment. The frequency of tdTomato+ cells among AP-eMBCs and GCBCs is shown for the indicated time points. Horizontal lines indicate the mean and error bars represent SD. Shown are representative results of two independent experiments.See also Figure S2.