Medical Biology - Research Publications
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ItemEpigenetic modulators of B cell fate identified through coupled phenotype-transcriptome analysis(SPRINGERNATURE, 2022-12)High-throughput methodologies are the cornerstone of screening approaches to identify novel compounds that regulate immune cell function. To identify novel targeted therapeutics to treat immune disorders and haematological malignancies, there is a need to integrate functional cellular information with the molecular mechanisms that regulate changes in immune cell phenotype. We facilitate this goal by combining quantitative methods for dissecting complex simultaneous cell phenotypic effects with genomic analysis. This combination strategy we term Multiplexed Analysis of Cells sequencing (MAC-seq), a modified version of Digital RNA with perturbation of Genes (DRUGseq). We applied MAC-seq to screen compounds that target the epigenetic machinery of B cells and assess altered humoral immunity by measuring changes in proliferation, survival, differentiation and transcription. This approach revealed that polycomb repressive complex 2 (PRC2) inhibitors promote antibody secreting cell (ASC) differentiation in both murine and human B cells in vitro. This is further validated using T cell-dependent immunization in mice. Functional dissection of downstream effectors of PRC2 using arrayed CRISPR screening uncovered novel regulators of B cell differentiation, including Mybl1, Myof, Gas7 and Atoh8. Together, our findings demonstrate that integrated phenotype-transcriptome analyses can be effectively combined with drug screening approaches to uncover the molecular circuitry that drives lymphocyte fate decisions.
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ItemThe concerted change in the distribution of cell cycle phases and zone composition in germinal centers is regulated by IL-21(NATURE PORTFOLIO, 2021-12-09)Humoral immune responses require germinal centres (GC) for antibody affinity maturation. Within GC, B cell proliferation and mutation are segregated from affinity-based positive selection in the dark zone (DZ) and light zone (LZ) substructures, respectively. While IL-21 is known to be important in affinity maturation and GC maintenance, here we show it is required for both establishing normal zone representation and preventing the accumulation of cells in the G1 cell cycle stage in the GC LZ. Cell cycle progression of DZ B cells is unaffected by IL-21 availability, as is the zone phenotype of the most highly proliferative GC B cells. Collectively, this study characterises the development of GC zones as a function of time and B cell proliferation and identifies IL-21 as an important regulator of these processes. These data help explain the requirement for IL-21 in normal antibody affinity maturation.
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ItemCyton2: A Model of Immune Cell Population Dynamics That Includes Familial Instructional Inheritance.(Frontiers Media SA, 2021)Lymphocytes are the central actors in adaptive immune responses. When challenged with antigen, a small number of B and T cells have a cognate receptor capable of recognising and responding to the insult. These cells proliferate, building an exponentially growing, differentiating clone army to fight off the threat, before ceasing to divide and dying over a period of weeks, leaving in their wake memory cells that are primed to rapidly respond to any repeated infection. Due to the non-linearity of lymphocyte population dynamics, mathematical models are needed to interrogate data from experimental studies. Due to lack of evidence to the contrary and appealing to arguments based on Occam's Razor, in these models newly born progeny are typically assumed to behave independently of their predecessors. Recent experimental studies, however, challenge that assumption, making clear that there is substantial inheritance of timed fate changes from each cell by its offspring, calling for a revision to the existing mathematical modelling paradigms used for information extraction. By assessing long-term live-cell imaging of stimulated murine B and T cells in vitro, we distilled the key phenomena of these within-family inheritances and used them to develop a new mathematical model, Cyton2, that encapsulates them. We establish the model's consistency with these newly observed fine-grained features. Two natural concerns for any model that includes familial correlations would be that it is overparameterised or computationally inefficient in data fitting, but neither is the case for Cyton2. We demonstrate Cyton2's utility by challenging it with high-throughput flow cytometry data, which confirms the robustness of its parameter estimation as well as its ability to extract biological meaning from complex mixed stimulation experiments. Cyton2, therefore, offers an alternate mathematical model, one that is, more aligned to experimental observation, for drawing inferences on lymphocyte population dynamics.
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ItemComparative evaluation of performance measures for shading correction in time-lapse fluorescence microscopy(WILEY, 2017-04)Time-lapse fluorescence microscopy is a valuable technology in cell biology, but it suffers from the inherent problem of intensity inhomogeneity due to uneven illumination or camera nonlinearity, known as shading artefacts. This will lead to inaccurate estimates of single-cell features such as average and total intensity. Numerous shading correction methods have been proposed to remove this effect. In order to compare the performance of different methods, many quantitative performance measures have been developed. However, there is little discussion about which performance measure should be generally applied for evaluation on real data, where the ground truth is absent. In this paper, the state-of-the-art shading correction methods and performance evaluation methods are reviewed. We implement 10 popular shading correction methods on two artificial datasets and four real ones. In order to make an objective comparison between those methods, we employ a number of quantitative performance measures. Extensive validation demonstrates that the coefficient of joint variation (CJV) is the most applicable measure in time-lapse fluorescence images. Based on this measure, we have proposed a novel shading correction method that performs better compared to well-established methods for a range of real data tested.
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ItemQuantifying NK cell growth and survival changes in response to cytokines and regulatory checkpoint blockade helps identify optimal culture and expansion conditions(WILEY, 2019-06)NK cells are innate lymphocytes critical for immune surveillance, particularly in eradication of metastatic cancer cells and acute antiviral responses. In contrast to T cells, NK cell-mediated immunity is rapid, with spontaneous cytotoxicity and cytokine/chemokine production upon pathogen detection. The renaissance in cancer immunology has cast NK cell biology back into the spotlight with an urgent need for deeper understanding of the regulatory networks that govern NK cell antitumor activity. To this end, we have adapted and refined a series of quantitative cellular calculus methods, previously applied to T and B lymphocytes, to dissect the biologic outcomes of NK cells following stimulation with cytokines (IL-15, IL-12, IL-18) or deletion of genes that regulate NK cell proliferation (Cish), survival (Bcl2l11), and activation-induced-cell-death (AICD; Fas). Our methodology is well suited to delineate effects on division rate, intrinsic apoptosis, and AICD, permitting variables such as population half-life, rate of cell division, and their combined influence on population numbers in response to stimuli to be accurately measured and modelled. Changes in these variables that result from gene deletion, concentration of stimuli, time, and cell density give insight into the dynamics of NK cell responses and serve as a platform to dissect the mechanism of action of putative checkpoints in NK cell activation and novel NK cell immunotherapy agents.
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ItemPre-mitotic genome re-organisation bookends the B cell differentiation process(NATURE RESEARCH, 2021-02-26)During cellular differentiation chromosome conformation is intricately remodelled to support the lineage-specific transcriptional programs required for initiating and maintaining lineage identity. When these changes occur in relation to cell cycle, division and time in response to cellular activation and differentiation signals has yet to be explored, although it has been proposed to occur during DNA synthesis or after mitosis. Here, we elucidate the chromosome conformational changes in B lymphocytes as they differentiate and expand from a naive, quiescent state into antibody secreting plasma cells. We find gene-regulatory chromosome reorganization in late G1 phase before the first division, and that this configuration is remarkably stable as the cells massively and rapidly clonally expand. A second wave of conformational change occurs as cells terminally differentiate into plasma cells, coincident with increased time in G1 phase. These results provide further explanation for how lymphocyte fate is imprinted prior to the first division. They also suggest that chromosome reconfiguration occurs prior to DNA replication and mitosis, and is linked to a gene expression program that controls the differentiation process required for the generation of immunity.
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ItemThe Rare Anaphylaxis-Associated FcγRIIa3 Exhibits Distinct Characteristics From the Canonical FcγRIIa1(FRONTIERS MEDIA SA, 2018-08-20)FcγRIIa is an activating FcγR, unique to humans and non-human primates. It induces antibody-dependent proinflammatory responses and exists predominantly as FcγRIIa1. A unique splice variant, we designated FcγRIIa3, has been reported to be associated with anaphylactic reactions to intravenous immunoglobulins (IVIg) therapy. We aim to define the functional consequences of this FcγRIIa variant associated with adverse responses to IVIg therapy and evaluate the frequency of associated SNPs. FcγRIIa forms from macaque and human PBMCs were investigated for IgG-subclass specificity, biochemistry, membrane localization, and functional activity. Disease-associated SNPs were analyzed by sequencing genomic DNA from 224 individuals with immunodeficiency or autoimmune disease. FcγRIIa3 was identified in macaque and human PBMC. The FcγRIIa3 is distinguished from the canonical FcγRIIa1 by a unique 19-amino acid cytoplasmic insertion and these two FcγRIIa forms responded distinctly to antibody ligation. Whereas FcγRIIa1 was rapidly internalized, FcγRIIa3 was retained longer at the membrane, inducing greater calcium mobilization and cell degranulation. Four FCGR2A SNPs were identified including the previously reported intronic SNP associated with anaphylaxis, but in only 1 of 224 individuals. The unique cytoplasmic element of FcγRIIa3 delays internalization and is associated with enhanced cellular activation. The frequency of the immunodeficiency-associated SNP varies between disease populations but interestingly occurred at a lower frequency than previously reported. None-the-less enhanced FcγRIIa3 function may promote a proinflammatory environment and predispose to pathological inflammatory responses.
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ItemModifying clonal selection theory with a probabilistic cell(WILEY, 2018-09)Problem-solving strategies in immunology currently utilize a series of ad hoc, qualitative variations on a foundation of Burnet's formulation of clonal selection theory. These modifications, including versions of two-signal theory, describe how signals regulate lymphocytes to make important decisions governing self-tolerance and changes to their effector and memory states. These theories are useful but are proving inadequate to explain the observable genesis and control of heterogeneity in cell types, the nonlinear passage of cell fate trajectories and how the input from multiple environmental signals can be integrated at different times and strengths. Here, I argue for a paradigm change to place immune theory on a firmer philosophical and quantitative foundation to resolve these difficulties. This change rejects the notion of identical cell subsets and substitutes the concept of a cell as comprised of autonomous functional mechanical components subject to stochastic variations in construction and operation. The theory aims to explain immunity in terms of cell population dynamics, dictated by the operation of cell machinery, such as randomizing elements, division counters, and fate timers. The effect of communicating signals alone and in combination within this system is determined with a cellular calculus. A series of models developed with these principles can resolve logical cell fate and signaling paradoxes and offer a reinterpretation for how self-non-self discrimination and immune response class are controlled.
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ItemA Model for Studying the Hemostatic Consumption or Destruction of Platelets(PUBLIC LIBRARY SCIENCE, 2013-03-07)A fundamental issue in understanding homeostasis of the hematopoietic system is to what extent intrinsic and extrinsic factors regulate cell fate. We recently revisited this issue for the case of blood platelets and concluded that platelet life span is largely regulated by internal factors, in contrast to the long-held view that accumulated damage from the environment triggers clearance. However, it is known that in humans there is an ongoing fixed requirement for platelets to maintain hemostasis and prevent bleeding; hence a proportion of platelets may be consumed in such processes before the end of their natural life span. Whether it is possible to detect this random loss of platelets in normal individuals at steady-state is unknown. To address this question, we have developed a mathematical model that independently incorporates age-independent random loss and age-dependent natural senescent clearance. By fitting to population survival curves, we illustrate the application of the model in quantifying the fixed requirement for platelets to maintain hemostasis in mice, and discuss the relationship with previous work in humans. Our results suggest a higher requirement for platelets in mice than in humans, however experimental uncertainty in the data limits our ability to constrain this quantity. We then explored the relationship between experimental uncertainty and parameter constraint using simulated data. We conclude that in order to provide useful constraint on the random loss fraction the standard error in the mean of the data must be reduced substantially, either through improving experimental uncertainty or increasing the number of experimental replicates to impractical levels. Finally we find that parameter constraint is improved at higher values of the random loss fraction; thus the model find utility in situations where the random loss fraction is expected to be high, for example during active bleeding or some types of thrombocytopenia.
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ItemQuantal and graded stimulation of B lymphocytes as alternative strategies for regulating adaptive immune responses(NATURE PUBLISHING GROUP, 2013-09)Lymphocytes undergo a typical response pattern following stimulation in vivo: they proliferate, differentiate to effector cells, cease dividing and predominantly die, leaving a small proportion of long-lived memory and effector cells. This pattern results from cell-intrinsic processes following activation and the influence of external regulation. Here we apply quantitative methods to study B-cell responses in vitro. Our results reveal that B cells stimulated through two Toll-like receptors (TLRs) require minimal external direction to undergo the basic pattern typical of immunity. Altering the stimulus strength regulates the outcome in a quantal manner by varying the number of cells that participate in the response. In contrast, the T-cell-dependent CD40 activation signal induces a response where division times and differentiation rates vary in relation to stimulus strength. These studies offer insight into how the adaptive antibody response may have evolved from simple autonomous response patterns to the highly regulable state that is now observed in mammals.
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