Figure 4. Primary CD40 signaling strength affects secondary B_mem cell differentiation, either to PCs or to GC B cells.

(a, b) Splenic B cells from each recipient mouse (containing iMB cells), generated as in Figure 3 (d), were cultured on 40LB feeder layers with IL-21 for 2 (a) or 3 (b) days. The expression of GL7 (a, b) and CD138 (b) on gated IgM⁺ or IgG1⁺ CD45.1⁺ (iMB cell-derived) cells was analyzed by FCM. (c–f) iMB-lo and iMB-hi cells were generated from B1-8 ki Igκ^−/− CD45.1/CD45.2 or B1-8 ki Igκ^−/− CD45.1 iGB cells, respectively, as in Figure 3 (d). 2.5 × 10⁴ (for ‘day 4’) or 1 × 10⁴ (for ‘day 10’) of NP⁺ iMB-lo and iMB-hi cells were mixed and co-transferred into WT B6 recipient mice with 1 × 10⁷ CGG-primed splenocytes. The recipient mice were then immunized with NP-CGG in alum and analyzed 4 or 10 days after immunization. (c) A schematic of the experimental procedure. (d) A representative FCM profile of the mixture of iMB-lo (CD45.2⁺) and iMB-hi (CD45.2⁻) cells, gated on CD45.1⁺ NP⁺ cells, before the transfer. (e) Representative FCM data at day 4 and day 10 after immunization showing the gating strategy. (f) The frequencies of iMB-lo- and iMB-hi-derived cells among CD45.1⁺ NP⁺ CD138⁻ or CD138⁺ cells at day 4, and among CD45.1⁺ NP⁺ B_mem cells (CD138⁻ GL7⁻ CD38⁺) or GC B cells (CD138⁻ GL7⁺ CD38⁻) at day 10. The mean of the values in each group is indicated by a horizontal bar (f). n.s., not significant (p>0.05); **, p<0.01; ****, p<0.0001; as determined by paired Student’s t tests (f). All data are representative of two independent experiments.

Figure 4—figure supplement 1. A gating strategy for IgM+ and IgG1+ cells used in Figure 4b.

Spleen cells containing donor-derived (CD45.1⁺) iMB cells (left) were cultured as shown in Figure 4a,b. FCM profiles of the gated iMB cell-derived cells on day 3, with the gating strategy for the cell fractions used in Figure 4b, are shown (right). The analysis on day 2 in Figure 4a was gated in the same manner.