A New Mechanism of Cell Cycle Progression Using C / N Ratio Measurements in Rosa26DHB-tdTomato Mice

Our model assumes that competition between LZ B cells is caused by the acquisition of a specific antigen from FDCs. The amount of help received for LZ B cells is proportional to their programmed number of divisions. Each division is accompanied by SHM with a probability of mutation (pmut ≈ 0.5)18, where mutations can be silent (psil = 0.5), lethal (plet = 0.3) (such as those that cause loss of B cell receptor (BCR) expression), affinity deleterious (pdel = 0.19) or affinity enhancing (penh = 0.01) on the basis of previously determined experimental and computational probabilities19,20,21. The cycle starts after division with DZ B cells returning to the LZ for further rounds of affinity-based selection. The parameters used in simulations are detailed in the Supplementary Table 1 and Methods.

We couldn’t track the B cells for long enough after the treatment ended, but we saw that the C/N ratios in D Z B cells remained above the threshold. 3l,m and Supplementary Videos 3 and 4). B cells being actively cycling didn’t enter a CDK2low G0 state.

The G0 and G1 phases of the cell cycle are characterized by low activity of cyclin-dependent kinase 2 (CDK2), which, when partnered with cyclin E, promotes progression through G1 and S-phase initiation43. To measure these phases precisely, we generated mice with a real-time reporter of CDK2 activity. 3b,c). This reporter is made up of human genes fused N-terminally to a red fluorescent tdTomato and inserted into the commonly expressedRosa26DHB. The DHB domain translocates from nucleus to cytoplasm after phosphorylation by CDK2. It therefore localizes to the nucleus when CDK2 activity is absent during G0, gradually exiting to the cytoplasm as cells progress through G1, S and G2 phases43,45 (Fig. 3b,d). The C /N ratio is used to determine the activity of CDK2 and can be used to monitor cell cycle progression. Rosa26DHB-tdTomato mice were crossed to the B1-8hi B cell receptor and a histone 2B (H2B)-EGFP reporter47 to accurately visualize mitotic events.

To track GC B cell division in vivo, we immunized H2b-mCherry mice with 4-hydroxy-3-nitrophenylacetyl conjugated to ovalbumin (NP-OVA), administered DOX on day 12.5 and assayed mCherry indicator dilution (Extended Data Fig. 2a,b). On the day 14 after DOX’s administration, mCherryhigh and mCherrylow represented 17% of GCB cells that had divided on an average of one or fewer times. There are 1c and 2b on this picture.

On the contribution of mutation-free clonal bursts to total GC response to DOX vaccinations: extended Data vs. experiment

The model predicts that a constant rate of around 1103 per base pair per cell division, equivalent to approximately 50% chance of mutations per division, will result in branched trees with only a few strands of the same sequence. 1c, black). Alternatively, the decreasing pmut model, with otherwise identical simulation parameters, predicted trees with nodes that extended up to 50 cells (Fig. 1c, red). Analysis of the experimental data revealed trees with nodes comprised of 2–15 members as well as trees with much larger nodes that contained 15–125 identical members (Fig. 2b and Extended Data Fig. 2f. The increasing pmut model could not account for the grossly expanded nodes of more than 15, but they were still consistent with the model.

After DOX exposure, profiling mCherryhigh and mCherrylow B cells can help compare cells that had divided one or fewer times. Many GCB cells were excluded from the analysis because they were mCherryintermediate. To assess the contribution of mutation-free clonal bursts to total GC responses, we performed single-cell analysis of all GC B cells responding to RBD vaccination (Extended Data Fig. 6a–c). The data obtained by mCherry was consistent with the results we observed when we observed large clusters of the same sequence. 6d–g. As expected, the grossly expanded nodes of B cells with more than 15 identical sequences also contributed disproportionately to the overall number of B cells and were highly enriched within high-affinity (RBD+) clones (Extended Data Fig. 6e–g. Thus, mutation-free clonal expansion contributes to affinity maturation in GC reactions across a range of immunological challenges.

We looked at if the constant pmut model could account for the large node observed in experimental data. To this end, we simulated the case with stochastic pmut (Extended Data Fig. There was a case where there was a constant amount of T cell help in the case of 7a. 7b). We found that neither scenario could account for the long-tail behaviour of node sizes observed in experiments. Thus, both the theory and experimental data are consistent with the idea that the per division rate of SHM is regulated and decreases with increasing T cell help.

AID introduces C>U Mutations at the preferential nucleotide sequence hotspots, with a highlighted R and A/G. To look into the possibility that there was a previous loss of these motifs in the expanded context, we profiled AID hotspots between cells. All of the cells were identical in appearance to the last one, and the data was extended to the last one. 8d–f. Thus, target motif decay does not account for differences in SHM between nodes. Finally, we compared the level of cell death among mCherry compartments and found no significant differences (Extended Data Fig. 8g,h).

The idea is that strong selection signals will decrease the DZ cells time in G0/G1 and reduce their exposure to AID.