Simon Mitchell

Rapid antibody production in response to invading pathogens requires the dramatic expansion of pathogen-derived antigen-specific B lymphocyte populations. Whether B cell population dynamics are based on stochastic competition between competing cell fates, as in the development of competence by the bacterium Bacillus sub-tilis, or on deterministic cell fate decisions that execute a predictable program, as during the development of the worm Caenorhabditis elegans, remains unclear. Here, we developed long-term live-cell microscopy of B cell population expansion and multiscale mechanis-tic computational modeling to characterize the role of molecular noise in determining phenotype heterogeneity. We show that the cell lineage trees underlying B cell population dynamics are mediated by a largely predictable decision-making process where the heterogeneity of cell proliferation and death decisions at any given timepoint largely derives from nongenetic heterogeneity in the founder cells. This means that contrary to previous models, only a minority of genetically identical founder cells contribute the majority to the population response. We computationally predict and experimentally confirm nongenetic molecular determinants that are pre-dictive of founder cells' proliferative capacity. While founder cell heterogeneity may arise from different exposure histories, we show that it may also be due to the gradual accumulation of small amounts of intrinsic noise during the lineage differentiation process of hematopoietic stem cells to mature B cells. Our finding of the largely deterministic nature of B lymphocyte responses may provide opportunities for diagnostic and therapeutic development. B cells | math model | heterogeneity | systems biology | proliferation H ow similar cells give rise to distinct fates is a fundamental question with different answers in different biological contexts. Cell fates are established deterministically for some cell types or organisms, such as Caenorhabditis elegans (1). In other cases, cell fates are established seemingly stochastically, such as for the development of competence by the bacterium Bacillus subtilis (2) or the generation of alternative color vision photore-ceptors in Drosophila melanogaster (3), and are thereby independent of cellular history (4). Here, we examined whether B lymphocyte proliferation decisions are the result of stochastic or deterministic fate decisions, and whether molecular network determinants may be identified. B lymphocytes are an essential component of the adaptive immune response and source of antibody-producing cells. In response to invading pathogens, B lymphocytes rapidly proliferate, differentiate into antibody-producing cells, and produce antigen-specific antibodies, which are essential for an effective immune response. B cells genetically diversify by rearranging the Ig locus to produce a diverse antibody repertoire and, therefore, diverse B cell receptor (BCR)-antigen affinities, which control mitogenic signals. While genetic heterogeneity arising from BCR diversification has the potential to be a source of heterogeneity of B cell fate, BCR-antigen affinity is a poor predictor of B cell proliferative expansion (5), and snapshot flow-cytometry measurements reveal a high degree of cell-to-cell generational heterogeneity even in response to BCR-independent stimuli (6). This led to the notion that B cell fate decision-making is highly stochastic. Indeed , direct measurement of division times at single-cell resolution revealed a highly variable first division (7, 8), consistent with a stochastic decision-making process. Based on these observations , Hodgkin et al. (9) developed a phenotypic model of lymphocyte proliferation using probability distributions of division and death times. The Cyton model has shown remarkable ability to fit dye dilution measurements by flow cytometry and derive corresponding cell biological parameters (such as division and death times) (9-13). Whereas a key assumption of the Cyton model is the independent stochastic decision-making of each cell at each generation, direct observation of sibling cell behavior revealed correlations in cell fate decisions and division times (8, 10, 11, 14). This has prompted revisions of the model to consider heritability. Thus, lymphocyte population dynamics models have been proposed that structure cell decisions by age (9, 15, 16) or division number (17) (or technical aspects; refs. 18 and 19). However, the degree to which fate decisions are nonstochastic remains unclear (20). Recently developed approaches combining multiple division-tracking dyes revealed that clonal populations were all of a similar generation at given time-points during the proliferative expansion phase (21). To mathematically account for these results, one recent study proposed a distributed division destiny time that is inherited through cell division , controlled in part by the proto-oncoprotein Myc and a separate "time-to-die" mechanism (22). Prior studies therefore provide the basis for considering the molecular mechanisms underlying B cell decision-making and, thereby, quantify the degree of inheritance versus intrinsic noise. Significance This study addresses why splenic B lymphocytes show differential cell proliferation and death decisions, and whether these may be predictable. Biology provides examples of both sto-chastic decision making and highly deterministic developmental programs. Prior studies of B lymphocytes suggested these cells make stochastic decisions, but the key experiment-long-term tracing of individual cell lineages-has not been done. Overcoming the technical challenges, we found that B cell fate decisions are largely nonstochastic. Using a mathematical model, we found that they are predictable, as long as the state of the molecular network in founder cells is known. That allowed us to identify the molecular determinants of proliferative fate decisions , which potentially constitute novel drug targets and biomarkers for B cell-mediated diseases.

Inflammation-responsive canonical NFκB induces many genes, two of which encode noncanonical NFκB pathway components that control developmental processes. This suggests potentially perilous cross-regulation by which inflammatory conditions could derail immune organ developmental decisions. We use mathematical modeling to propose a mechanism that functions as a brake on this connection. We report that the key enzyme mediating developmental NFκB is subject to competition from two forms of its single substrate. Termed substrate complex competition, this regulatory motif can lead to a counterintuitive decrease of signaling product in conditions of elevated substrate abundance. We propose that although noncanonical NFκB requires intact inflammatory NFκB signaling, substrate complex competition allows developmental signals to be reliably transduced without inappropriate amplification by inflammation.