Cellular proliferation is an essential feature of the adaptive immune response. The introduction of the division tracking dye carboxyfluorescein diacetate succinimidyl ester (CFSE) has made it possible to monitor the number of cell divisions during proliferation and to examine the relationship between proliferation and differentiation. Although qualitative examination of CFSE data may be useful, substantially more information about division and death rates can be extracted from quantitative CFSE time-series experiments. Quantitative methods can reveal in detail how lymphocyte proliferation and survival are regulated and altered by signals such as those received from co-stimulatory molecules, drugs and genetic polymorphisms. In this protocol, we present a detailed method for examining time-series data using graphical and computer-based procedures available to all experimenters.

In contrast to most stimulated lymphocytes, B cells exposed to Toll-like receptor 9 ligands are nonself-adherent, allowing individual cells and families to be followed in vitro for up to 5 days. These B cells undergo phases typical of an adaptive response, dividing up to 6 times before losing the impetus for further growth and division and eventually dying by apoptosis. Using long-term microscopic imaging, accurate histories of individual lymphocyte fates were collected. Quantitative analysis of family relationships revealed that times to divide of siblings were strongly related but these correlations were progressively lost through consecutive divisions. A weaker, but significant, correlation was also found for death times among siblings. Division cessation is characterized by a loss of cell growth and the division in which this occurs is strongly inherited from the original founder cell and is related to the size this cell reaches before its first division. Thus, simple division-based dilution of factors synthesized during the first division may control the maximum division reached by stimulated cells. The stochastic distributions of times to divide, times to die, and divisions reached are also measured. Together, these results highlight the internal cellular mechanisms that control immune responses and provide a foundation for the development of new mathematical models that are correct at both single-cell and population levels. The coordinated regulation of cell proliferation and apoptosis is a striking feature of the adaptive response of lymphocytes after exposure to pathogens. The initially quiescent lymphocytes are stimulated to undergo a series of proliferation cycles that increase the number of reactive cells many hundredfold. After a period the response peaks, cells stop dividing, and Ϸ95% of the newly generated cells die by apoptosis (1, 2). Although these population kinetics are well understood, we have little knowledge of how the component single-cell fates add up to this outcome, nor how individual decisions of division, death, and quiescence are handled. Our current models of lymphocyte responses are strongly influenced by studies of tumor cells and fibroblasts undertaken by investigators in the 1960s and 1970s. At that time film and microscopy were used to measure the kinetics of cell division in vitro (3-5). These studies noted that intermitotic division times were different between cell types and that all eukaryotic cells, including both yeast and clonally derived tumor cell populations, exhibited significant variation within the population of dividing cells.The variation observed in cell cycle times was incorporated into the widely used Smith and Martin mathematical model of cell growth that postulated a random time spent in an ''A state'' (assumed to be G 1 ) that governed entry into a deterministic B phase (S, G 2 , and M) of the cell cycle (6, 7). Because the Smith-Martin model is relatively easy to implement, it has served as the backbone for numerous models of lymphocyte proliferation (6-1...

In response to stimulation, B lymphocytes pursue a large number of distinct fates important for immune regulation. Whether each cell's fate is determined by external direction, internal stochastic processes, or directed asymmetric division is unknown. Measurement of times to isotype switch, to develop into a plasmablast, and to divide or to die for thousands of cells indicated that each fate is pursued autonomously and stochastically. As a consequence of competition between these processes, censorship of alternative outcomes predicts intricate correlations that are observed in the data. Stochastic competition can explain how the allocation of a proportion of B cells to each cell fate is achieved. The B cell may exemplify how other complex cell differentiation systems are controlled.

Problems can arise when vaccines and wild strains of a chicken herpesvirus recombine.

Stochastic variation in cell cycle time is a consistent feature of otherwise similar cells within a growing population. Classic studies concluded that the bulk of the variation occurs in the G 1 phase, and many mathematical models assume a constant time for traversing the S/G 2 /M phases. By direct observation of transgenic fluorescent fusion proteins that report the onset of S phase, we establish that dividing B and T lymphocytes spend a near-fixed proportion of total division time in S/G 2 /M phases, and this proportion is correlated between sibling cells. This result is inconsistent with models that assume independent times for consecutive phases. Instead, we propose a stretching model for dividing lymphocytes where all parts of the cell cycle are proportional to total division time. Data fitting based on a stretched cell cycle model can significantly improve estimates of cell cycle parameters drawn from DNA labeling data used to monitor immune cell dynamics.he kinetic relationship between phases of the cell cycle first came to attention with the advent of autoradiographic techniques for detecting DNA synthesis in the 1950s (1, 2). It was realized that such data could be used to resolve the dynamics of the proliferating population if combined with an appropriate cell cycle model. However, direct filming of times to divide revealed remarkable variation, even among cloned, presumed identical, cells (3-6), eliminating simple deterministic models as the basis for cell cycle control. Working toward developing a general model, Smith and Martin made the striking observation that plotting the proportion of undivided cells versus time (so-called "alpha plots"), gave curves suggestive of two distinct phases, one relatively constant and another stochastic (7). They proposed that the two phases mapped to discrete states of the cell cycle. A resting "A state," they suggested, was contained within the G 1 phase from which cells could exit with constant probability per unit time (analogous to radioactive decay). The cells then entered the "B phase," which includes that part of G 1 not included in A state, as well as the entirety of S/G 2 /M. In B phase, cells' activities were first described to be "deterministic, and directed towards replication," implying a constant B phase. However, in the same paper, this assumption was relaxed and the duration of B phase was described with a relatively constant random variable (7).Although details of the quantitative relationship and biological interpretation have been debated (7-12), the rule that the bulk of kinetic variation is in G 1 phase, and that time in S/G 2 /M is relatively fixed, is widely accepted. Furthermore, mathematical models adopting this mechanical description (so-called "transition probability" or "compartment" models) remain popular and form the basis of many studies of lymphocyte and cancer kinetics in vitro and in vivo today (13-21).More recently, a molecular description of cell cycle regulation, including the discovery of key regulatory proteins such as cyclins a...

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Key Points • The first embryonic platelets are produced by a unique lineage of diploid cells not polyploid MKs. • Diploid platelet-forming cells are produced in the early mouse embryo via a progenitor cell-independent pathway. In this study, we test the assumption that the hematopoietic progenitor/colony-forming cells of the embryonic yolk sac (YS), which are endowed with megakaryocytic potential, differentiate into the first platelet-forming cells in vivo. We demonstrate that from embryonic day (E) 8.5 all megakaryocyte (MK) colony-forming cells belong to the conventional hematopoietic progenitor cell (HPC) compartment. Although these cells are indeed capable of generating polyploid MKs, they are not the source of the first platelet-forming cells. We show that proplatelet formation first occurs in a unique and previously unrecognized lineage of diploid platelet-forming cells, which develop within the YS in parallel to HPCs but can be specified in the E8.5 Runx1-null embryo despite the absence of the progenitor cell lineage. (Blood. 2014;124(17):2725-2729)

In contrast to the RNA viruses, the genome of large DNA viruses such as herpesviruses have been considered to be relatively stable. Intra-specific recombination has been proposed as an important, but underestimated, driving force in herpesvirus evolution. Recently, two distinct field strains of infectious laryngotracheitis virus (ILTV) have been shown to have arisen from independent recombination events between different commercial ILTV vaccines. In this study we sequenced the genomes of additional ILTV strains and also utilized other recently updated complete genome sequences of ILTV to confirm the existence of a number of ILTV recombinants in nature. Multiple recombination events were detected in the unique long and repeat regions of the genome, but not in the unique short region. Most recombinants contained a pair of crossover points between two distinct lineages of ILTV, corresponding to the European origin and the Australian origin vaccine strains of ILTV. These results suggest that there are two distinct genotypic lineages of ILTV and that these commonly recombine in the field.