Michal Or-Guil

Recirculation of germinal center B cells: a multilevel selection strategy for antibody maturation.

Summary:  Optimization of antibody affinity is a hallmark of the humoral immune response. It takes place in hundreds of transient microstructures called germinal centers (GCs). Their function and time‐dependent behavior are subjects of active investigation. According to a generally accepted notion, their individual kinetics follows the average kinetics of all GCs present in the observed lymphatic tissue. In this review, we challenge this view and point out, with the help of mathematical simulations, that inferring the kinetics of individual GCs from cross‐sectional evaluation of GC kinetics is virtually impossible. Thus, the time course of individual GCs is open to conjecture. For instance, one possible interpretation is that GCs exist for a time span considerably shorter than that of the observed average kinetics. We explore the implications of different temporal organizations of GCs in the light of the hypothesis that GC B‐cell emigrants recolonize GC niches. This assumption leads to a view where GCs work in parallel but are linked by recirculation of B‐cell emigrants. In this view, interleaved global and local competition provide for an implementation of multiple levels of B‐cell selection in affinity maturation. The concepts of iteration, all‐or‐none behavior, and phasic mutation schedule are discussed in the light of this hypothesis.

Stable bound states of pulses in an excitable medium

Abstract The interaction of one-dimensional pulses is studied in the excitable regime of a two variable reaction–diffusion model. The model is capable of exhibiting long range attraction of pulses and formation of stable bound pulse states. The important features of pulse interactions can be captured by a combination of various analytical and numerical methods. A kinematic ansatz treating pulses as particle-like interacting structures is described. Their interaction is determined using the dispersion relation for pulse trains, which gives the dependence of the speed c ( d ) of the wavetrain on its wavelength d. Anomalous dispersion for large d, i.e. a negative slope of c ( d ), corresponds to long range pulse attraction. Stable bound pairs are possible if the medium exhibits long range attraction and there is at least one maximum of the dispersion curve. We compare predictions of the kinematic theory with numerical simulations and stability analysis. If the slope of the dispersion curve changes sign, branches of non-equidistant pulse train solutions bifurcate and may lead to bound pulse states. The transition from normal long range dispersion, typical in excitable media, to the anomalous dispersion studied here can be understood through a multiscale perturbation theory for pulse interactions. We derive the relevant equations, which yield an analytic expression for non-monotonic dispersion curves with a finite number of extrema.

A “Crossomics” Study Analysing Variability of Different Components in Peripheral Blood of Healthy Caucasoid Individuals

Background Different immunotherapy approaches for the treatment of cancer and autoimmune diseases are being developed and tested in clinical studies worldwide. Their resulting complex experimental data should be properly evaluated, therefore reliable normal healthy control baseline values are indispensable. Methodology/Principal Findings To assess intra- and inter-individual variability of various biomarkers, peripheral blood of 16 age and gender equilibrated healthy volunteers was sampled on 3 different days within a period of one month. Complex “crossomics” analyses of plasma metabolite profiles, antibody concentrations and lymphocyte subset counts as well as whole genome expression profiling in CD4+T and NK cells were performed. Some of the observed age, gender and BMI dependences are in agreement with the existing knowledge, like negative correlation between sex hormone levels and age or BMI related increase in lipids and soluble sugars. Thus we can assume that the distribution of all 39.743 analysed markers is well representing the normal Caucasoid population. All lymphocyte subsets, 20% of metabolites and less than 10% of genes, were identified as highly variable in our dataset. Conclusions/Significance Our study shows that the intra-individual variability was at least two-fold lower compared to the inter-individual one at all investigated levels, showing the importance of personalised medicine approach from yet another perspective.

Is There a Typical Germinal Center? A Large-Scale Immunohistological Study on the Cellular Composition of Germinal Centers during the Hapten-Carrier–Driven Primary Immune Response in Mice

Germinal centers (GCs) are complex, multicell-type, transient structures that form in secondary lymphatic tissues in response to T cell-dependent stimulation. This process is crucial to the adaptive immune response because it is the source of affinity maturation and long-lived B cell memory. Our previous studies showed that the growth of murine splenic GCs is nonsynchronized, involving broad-volume distributions of individual GCs at any time. This raises the question whether such a thing as a typical GC exists. To address this matter, we acquired large-scale confocal data on GCs throughout the course of the 2-phenyl-5-oxazolone chicken serum albumin-driven primary immune response in BALB/c mice. Semiautomated image analysis of 3457 GC sections revealed that, although there is no typical GC in terms of size, GCs have a typical cellular composition in that the cell ratios of resident T cells, macrophages, proliferating cells, and apoptotic nuclei are maintained during the established phase of the response. Moreover, our data provide evidence that the dark zone (DZ) and light zone (LZ) compartments of GCs are about the same size and led us to estimate that the minimal cell loss rate in GCs is 3% per hour. Furthermore, we found that the population of GC macrophages is larger and more heterogeneous than previously thought, and that despite enrichment of T cells in the LZ, the DZ of murine splenic GCs is not poor in T cells. DZ and LZ differ in the T cell-to-macrophage ratio rather than in the density of T cells.

Quantifying the Length and Variance of the Eukaryotic Cell Cycle Phases by a Stochastic Model and Dual Nucleoside Pulse Labelling

A fundamental property of cell populations is their growth rate as well as the time needed for cell division and its variance. The eukaryotic cell cycle progresses in an ordered sequence through the phases and and is regulated by environmental cues and by intracellular checkpoints. Reflecting this regulatory complexity, the length of each phase varies considerably in different kinds of cells but also among genetically and morphologically indistinguishable cells. This article addresses the question of how to describe and quantify the mean and variance of the cell cycle phase lengths. A phase-resolved cell cycle model is introduced assuming that phase completion times are distributed as delayed exponential functions, capturing the observations that each realization of a cycle phase is variable in length and requires a minimal time. In this model, the total cell cycle length is distributed as a delayed hypoexponential function that closely reproduces empirical distributions. Analytic solutions are derived for the proportions of cells in each cycle phase in a population growing under balanced growth and under specific non-stationary conditions. These solutions are then adapted to describe conventional cell cycle kinetic assays based on pulse labelling with nucleoside analogs. The model fits well to data obtained with two distinct proliferating cell lines labelled with a single bromodeoxiuridine pulse. However, whereas mean lengths are precisely estimated for all phases, the respective variances remain uncertain. To overcome this limitation, a redesigned experimental protocol is derived and validated in silico. The novelty is the timing of two consecutive pulses with distinct nucleosides that enables accurate and precise estimation of both the mean and the variance of the length of all phases. The proposed methodology to quantify the phase length distributions gives results potentially equivalent to those obtained with modern phase-specific biosensor-based fluorescent imaging.

Fold-Hopf bursting in a model for calcium signal transduction

We study a recent model for calcium signal transduction. This model displays spiking, bursting and chaotic oscillations in accordance with experimental results. We calculate bifurcation diagrams and study the bursting behaviour in detail. This behaviour is classified according to the dynamics of separated slow and fast subsystems. It is shown to be of the Fold–Hopf type, a type which was previously only described in the context of neuronal systems, but not in the context of signal transduction in the cell.

Oscillations of fronts and front pairs in two- and three-component reaction-diffusion systems

Abstract We investigate multi-co,ponent reaction-diffusion systems on a finite one-dimensional domain. The first component is assumed both to react on a short time-scale, and to have a small diffusion length leading to patterns with sharp fronts. To analyze stability, we apply the SLEP method by Nishiura and Fujii. This method is superior to standard perturbation methods. We illustrate this using a two-component system. In the three-component system, in a situation where both the influence of the first component on the other components and the interaction of the latter is only weak, we show that there is a unique Hopf destabilization of stationary fronts when changing time constants. As an example we treat a system with global coupling used e.g. for semi-conductor devices. For filaments (front pairs) we find two types of Hopf destabilizations: breathing and swinging. The global coupling controls which type occurs. This corresponds to recent experimental results and is confirmed via numerical calculations.

Broad Volume Distributions Indicate Nonsynchronized Growth and Suggest Sudden Collapses of Germinal Center B Cell Populations

Immunization with a T cell-dependent Ag leads to the formation of several hundred germinal centers (GCs) within secondary lymphoid organs, a key process in the maturation of the immune response. Although prevailing perceptions about affinity maturation intuitively assume simultaneous seeding, growth, and decay of GCs, our previous mathematical simulations led us to hypothesize that their growth might be nonsynchronized. To investigate this, we performed computer-aided three-dimensional reconstructions of splenic GCs to measure size distributions at consecutive time points following immunization of BALB/c mice with a conjugate of 2-phenyl-oxazolone and chicken serum albumin. Our analysis reveals a broad volume distribution of GCs, indicating that individual GCs certainly do not obey the average time course of the GC volumes and that their growth is nonsynchronized. To address the cause and implications of this behavior, we compared our empirical data with simulations of a stochastic mathematical model that allows for frequent and sudden collapses of GCs. Strikingly, this model succeeds in reproducing the empirical average kinetics of GC volumes as well as the underlying broad size distributions. Possible causes of GC B cell population collapses are discussed in the context of the affinity-maturation process.

Epitope Mapping Using Randomly Generated Peptide Libraries

Characterizing the immune response towards a pathogen is of high interest for vaccine development and diagnosis. However, the characterization of disease-related antigen-antibody interactions is of enormous complexity. Here, we present a method comprising binding studies of serum antibody pools to synthetic random peptide libraries, and data analysis of the resulting binding patterns. The analysis can be applied to classify and predict different groups of individuals and to detect the peptides which best discriminate the investigated groups. As an example, the analysis of antibody repertoire binding patterns of different mice strains and of mice infected with helminth parasites is shown. Due to the design of the library and the sophisticated analysis, the method is able to classify and predict the different mice strains and the infection with very high accuracy and with a very small number of peptides, illustrating the potential of random library screenings in determining molecular markers for diagnosis.

Affinity maturation of B cells involves not only a few but a whole spectrum of relevant mutations

Affinity maturation of B lymphocytes within germinal centers involves both diversification of their B-cell receptors (BCRs) by somatic hypermutation (SHM) and a crucial receptor-mediated selection step. However, in contrast to recent advances in revealing the molecular mechanism of SHM, the fundamentals of the selection process are still poorly understood, i.e. it is often not clear how and how many mutations contribute to improving a BCR during the response against a given antigen. A general drawback in assessing the mutations relevant to the selection process is the difficult task of rating the relative contributions of selection and intrinsic biases to the experimentally observed mutation patterns of BCRs. The approach proposed here is premised on statistical comparison of the frequency distributions of nucleotide substitutions as observed in datasets of hypermutated BCRs against their frequency distribution expected under the null hypothesis of no selection. Thereby, we show that the spectrum of mutations relevant to maturation of canonical anti-(4-hydroxy-3-nitrophenyl)acetyl BCRs is much broader than previously acknowledged, going beyond the scope of single key mutations. Moreover, our results suggest that maturation not only involves selection by means of affinity but likewise expression and stabilization of BCRs.