Lineage tracing reveals B cell antibody class switching is stochastic, cell-autonomous, and tuneable.

To optimize immunity to pathogens, B lymphocytes generate plasma cells with functionally diverse antibody isotypes. By lineage tracing single cells within differentiating B cell clones, we identified the heritability of discrete fate controlling mechanisms to inform a general mathematical model of B cell fate regulation. Founder cells highly influenced clonal plasma-cell fate, whereas class switch recombination (CSR) was variegated within clones. In turn, these CSR patterns resulted from independent all-or-none expression of both activation-induced cytidine deaminase (AID) and IgH germline transcription (GLT), with the latter being randomly re-expressed after each cell division. A stochastic model premised on these molecular transition rules accurately predicted antibody switching outcomes under varied conditions in vitro and during an immune response in vivo. Thus, the generation of functionally diverse antibody types follows rules of autonomous cellular programming that can be adapted and modeled for the rational control of antibody classes for potential therapeutic benefit.

A Myc-dependent division timer complements a cell-death timer to regulate T cell and B cell responses.

T lymphocytes and B lymphocytes integrate activating signals to control the size of their proliferative response. Here we report that such control was achieved by timed changes in the production rate of cell-cycle-regulating proto-oncoprotein Myc, with division cessation occurring when Myc levels fell below a critical threshold. The changing pattern of the level of Myc was not affected by cell division, which identified the regulating mechanism as a cell-intrinsic, heritable temporal controller. Overexpression of Myc in stimulated T cells and B cells did not sustain cell proliferation indefinitely, as a separate 'time-to-die' mechanism, also heritable, was programmed after lymphocyte activation and led to eventual cell loss. Together the two competing cell-intrinsic timed fates created the canonical T cell and B cell immune-response pattern of rapid growth followed by loss of most cells. Furthermore, small changes in these timed processes by regulatory signals, or by oncogenic transformation, acted in synergy to greatly enhance cell numbers over time.

Programmed death receptor 1 (PD-1) ligand Fc fusion proteins reduce T-cell proliferation in vitro independently of PD-1.

Programmed death receptor 1 (PD-1) is an inhibitory receptor on T cells shown to restrain T-cell proliferation. PD-1 immune checkpoint blockade has emerged as a highly promising approach in cancer treatment. Much of our understanding of the function of PD-1 is derived from in vitro T-cell activation assays. Here we set out to further investigate how T cells integrate inhibitory signals such as PD-1 in vitro using the PD-1 agonist, PD-1 ligand 1 (PD-L1) fusion protein (PD-L1.Fc), coimmobilized alongside anti-CD3 agonist monoclonal antibody (mAb) on plates to deliver PD-1 signals to wild-type and PD-1-/- CD8+ T cells. Surprisingly, we found that the PD-L1.Fc fusion protein inhibited T-cell proliferation independently of PD-1. This PD-L1.Fc inhibition was observed in the presence and absence of CD28 and interleukin-2 signaling. Binding of PD-L1.Fc was restricted to PD-1-expressing T cells and thus inhibition was not mediated by the interaction of PD-L1.Fc with CD80 or other yet unknown binding partners. Furthermore, a similar PD-1-independent reduction of T-cell proliferation was observed with plate-bound PD-L2.Fc. Hence, our results suggest that the coimmobilization of PD-1 ligand fusion proteins with anti-CD3 mAb leads to a reduction of T-cell engagement with plate-bound anti-CD3 mAb. This study demonstrates a nonspecific mechanism of T-cell inhibition when PD-L1.Fc or PD-L2.Fc fusion proteins are delivered in a plate-bound coimmobilization assay and highlights the importance of careful optimization of assay systems and reagents when interpreting their influence on T-cell proliferation.

Survival and division fate programs are preserved but retuned during the naïve to memory CD8+ T-cell transition.

Memory T cells are generated from naïve precursors undergoing proliferation during the initial immune response. Both naïve and memory T cells are maintained in a resting, quiescent state and respond to activation with a controlled proliferative burst and differentiation into effector cells. This similarity in the maintenance and response dynamics points to the preservation of key cellular fate programs; however, whether memory T cells have acquired intrinsic changes in these programs that may contribute to the enhanced immune protection in a recall response is not fully understood. Here we used a quantitative model-based analysis of proliferation and survival kinetics of in vitro-stimulated murine naïve and memory CD8+ T cells in response to homeostatic and activating signals to establish intrinsic similarities or differences within these cell types. We show that resting memory T cells display heightened sensitivity to homeostatic cytokines, responding to interleukin (IL)-2 in addition to IL-7 and IL-15. The proliferative response to αCD3 was equal in size and kinetics, demonstrating that memory T cells undergo the same controlled division burst and automated return to quiescence as naïve T cells. However, perhaps surprisingly, we observed reduced expansion of αCD3-stimulated memory T cells in response to activating signals αCD28 and IL-2 compared with naïve T cells. Overall, we demonstrate that although sensitivities to cytokine and costimulatory signals have shifted, fate programs regulating the scale of the division burst are conserved in memory T cells.

Chromosome social distancing and crowd control: the dual role of Ki67.

The Ki67 protein is proposed to have two conformations; one which segregates chromosomes before anaphase, and the other which results in chromosome condensation after cell division to exclude large cytosolic components from the reforming nuclei of daughter cells.

Speculations on the evolution of humoral adaptive immunity.

The protection of a multicellular organism from infection, at both cell and humoral levels, has been a tremendous driver of gene selection and cellular response strategies. Here we focus on a critical event in the development of humoral immunity: The transition from principally innate responses to a system of adaptive cell selection, with all the attendant mechanical problems that must be solved in order for it to work effectively. Here we review recent advances, but our major goal is to highlight that the development of adaptive immunity resulted from the adoption, reuse and repurposing of an ancient, autonomous cellular program that combines and exploits three titratable cellular fate timers. We illustrate how this common cell machinery recurs and appears throughout biology, and has been essential for the evolution of complex organisms, at many levels of scale.

Multiplexed Division Tracking Dyes for Proliferation-Based Clonal Lineage Tracing.

The generation of cellular heterogeneity is an essential feature of immune responses. Understanding the heritability and asymmetry of phenotypic changes throughout this process requires determination of clonal-level contributions to fate selection. Evaluating intraclonal and interclonal heterogeneity and the influence of distinct fate determinants in large numbers of cell lineages, however, is usually laborious, requiring familial tracing and fate mapping. In this study, we introduce a novel, accessible, high-throughput method for measuring familial fate changes with accompanying statistical tools for testing hypotheses. The method combines multiplexing of division tracking dyes with detection of phenotypic markers to reveal clonal lineage properties. We illustrate the method by studying in vitro-activated mouse CD8+ T cell cultures, reporting division and phenotypic changes at the level of families. This approach has broad utility as it is flexible and adaptable to many cell types and to modifications of in vitro, and potentially in vivo, fate monitoring systems.

German Society for Immunology and Australasian Society for Immunology joint Workshop 3(rd) -4(th) December 2015 - Meeting report.

The German Society for Immunology (DGfI) and the Australasian Society for Immunology (ASI) hosted the first DGfI-ASI joint workshop from December 3-4, 2015 in Canberra, Australia. A delegation of 15 distinguished German immunologists discussed the workshop topic "immune regulation in infections and immune mediated diseases" with the aim to establish new German-Australasian collaborations, discuss new concepts in the field of immune regulation and build a scientific network to create more utilizable resources for excellent (trans-border) immunological research. The workshop was associated with the 45(th) Annual Scientific Meeting of the ASI held from Nov 29-Dec 3, 2015, opening up even more opportunities for finding new collaboration partners. A return meeting will be linked to the annual DGfI meeting that will take place in 2017 in Erlangen.

Stretched cell cycle model for proliferating lymphocytes.

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 G1 phase, and many mathematical models assume a constant time for traversing the S/G2/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/G2/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.

Genome-wide CRISPR screening identifies a role for ARRDC3 in TRP53-mediated responses.

Whole-genome screens using CRISPR technologies are powerful tools to identify novel tumour suppressors as well as factors that impact responses of malignant cells to anti-cancer agents. Applying this methodology to lymphoma cells, we conducted a genome-wide screen to identify novel inhibitors of tumour expansion that are induced by the tumour suppressor TRP53. We discovered that the absence of Arrestin domain containing 3 (ARRDC3) increases the survival and long-term competitiveness of MYC-driven lymphoma cells when treated with anti-cancer agents that activate TRP53. Deleting Arrdc3 in mice caused perinatal lethality due to various developmental abnormalities, including cardiac defects. Notably, the absence of ARRDC3 markedly accelerated MYC-driven lymphoma development. Thus, ARRDC3 is a new mediator of TRP53-mediated suppression of tumour expansion, and this discovery may open new avenues to harness this process for cancer therapy.

Cyton2: A Model of Immune Cell Population Dynamics That Includes Familial Instructional Inheritance.

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.

Differential expansion of T central memory precursor and effector subsets is regulated by division speed.

While antigen-primed T cells proliferate at speeds close to the physiologic maximum of mammalian cells, T cell memory is maintained in the absence of antigen by rare cell divisions. The transition between these distinct proliferative programs has been difficult to resolve via population-based analyses. Here, we computationally reconstruct the proliferative history of single CD8+ T cells upon vaccination and measure the division speed of emerging T cell subsets in vivo. We find that slower cycling central memory precursors, characterized by an elongated G1 phase, segregate early from the bulk of rapidly dividing effector subsets, and further slow-down their cell cycle upon premature removal of antigenic stimuli. In contrast, curtailed availability of inflammatory stimuli selectively restrains effector T cell proliferation due to reduced receptivity for interleukin-2. In line with these findings, persistence of antigenic but not inflammatory stimuli throughout clonal expansion critically determines the later size of the memory compartment.

Temporal Analysis of Brd4 Displacement in the Control of B Cell Survival, Proliferation, and Differentiation.

JQ1 is a BET-bromodomain inhibitor that has immunomodulatory effects. However, the precise molecular mechanism that JQ1 targets to elicit changes in antibody production is not understood. Our results show that JQ1 induces apoptosis, reduces cell proliferation, and as a consequence, inhibits antibody-secreting cell differentiation. ChIP-sequencing reveals a selective displacement of Brd4 in response to acute JQ1 treatment (<2 h), resulting in specific transcriptional repression. After 8 h, subsequent alterations in gene expression arise as a result of the global loss of Brd4 occupancy. We demonstrate that apoptosis induced by JQ1 is solely attributed to the pro-apoptotic protein Bim (Bcl2l11). Conversely, cell-cycle regulation by JQ1 is associated with multiple Myc-associated gene targets. Our results demonstrate that JQ1 drives temporal changes in Brd4 displacement that results in a specific transcriptional profile that directly affects B cell survival and proliferation to modulate the humoral immune response.

The regulation of lymphocyte activation and proliferation.

Activation induced proliferation and clonal expansion of antigen specific lymphocytes is a hallmark of the adaptive immune response to pathogens. Recent studies identify two distinct control phases. In the first T and B lymphocytes integrate antigen and additional costimuli to motivate a programmed proliferative burst that ceases with a return to cell quiescence and eventual death. This proliferative burst is autonomously timed, ensuring an appropriate response magnitude whilst preventing uncontrolled expansion. This initial response is subject to further modification and extension by a range of signals that modify, expand and direct the emergence of a rich array of new cell types. Thus, both robust clonal expansion of a small number of antigen specific T cells, and the concurrent emergence of extensive cellular diversity, confers immunity to a vast array of different pathogens. The in vivo response to a given pathogen is made up by the sum of all responding clones and is reproducible and pathogen specific. Thus, a precise description of the regulatory principles governing lymphocyte proliferation, differentiation and survival is essential to a unified understanding of the immune system.

Regulatory T Cells Suppress Effector T Cell Proliferation by Limiting Division Destiny.

Understanding how the strength of an effector T cell response is regulated is a fundamental problem in immunology with implications for immunity to pathogens, autoimmunity, and immunotherapy. The initial magnitude of the T cell response is determined by the sum of independent signals from antigen, co-stimulation and cytokines. By applying quantitative methods, the contribution of each signal to the number of divisions T cells undergo (division destiny) can be measured, and the resultant exponential increase in response magnitude accurately calculated. CD4+CD25+Foxp3+ regulatory T cells suppress self-reactive T cell responses and limit pathogen-directed immune responses before bystander damage occurs. Using a quantitative modeling framework to measure T cell signal integration and response, we show that Tregs modulate division destiny, rather than directly increasing the rate of death or delaying interdivision times. The quantitative effect of Tregs could be mimicked by modulating the availability of stimulatory co-stimuli and cytokines or through the addition of inhibitory signals. Thus, our analysis illustrates the primary effect of Tregs on the magnitude of effector T cell responses is mediated by modifying division destiny of responding cell populations.

Life and Death of Activated T Cells: How Are They Different from Naïve T Cells?

T cells are pivotal in immunity and immunopathology. After activation, T cells undergo a clonal expansion and differentiation followed by a contraction phase, once the pathogen has been cleared. Cell survival and cell death are critical for controlling the numbers of naïve T cells, effector, and memory T cells. While naïve T cell survival has been studied for a long time, more effort has gone into understanding the survival and death of activated T cells. Despite this effort, there is still much to be learnt about T cell survival, as T cells transition from naïve to effector to memory. One key advance is the development of inhibitors that may allow the temporal study of survival mechanisms operating in these distinct cell states. Naïve T cells were highly reliant on BCL-2 and sensitive to BCL-2 inhibition. Activated T cells are remarkably different in their regulation of apoptosis by pro- and antiapoptotic members of the BCL-2 family, rendering them differentially sensitive to antagonists blocking the function of one or more members of this family. Recent progress in understanding other programmed cell death mechanisms, especially necroptosis, suggests a unique role for alternative pathways in regulating death of activated T cells. Furthermore, we highlight a mechanism of epigenetic regulation of cell survival unique to activated T cells. Together, we present an update of our current understanding of the survival requirement of activated T cells.

Characterisation of mice lacking all functional isoforms of the pro-survival BCL-2 family member A1 reveals minor defects in the haematopoietic compartment.

The pro-survival proteins of the BCL-2 family regulate the survival of all cells, and genetic deletion models for these proteins have revealed which specific BCL-2 family member(s) is/are critical for the survival of particular cell types. A1 is a pro-survival BCL-2-like protein that is expressed predominantly in haematopoietic cells, and here we describe the characterisation of a novel mouse strain that lacks all three functional isoforms of A1 (A1-a, A1-b and A1-d). Surprisingly, complete loss of A1 caused only minor defects, with significant, although relatively small, decreases in γδTCR T cells, antigen-experienced conventional as well as regulatory CD4 T cells and conventional dendritic cells (cDCs). When examining these cell types in tissue culture, only cDC survival was significantly impaired by the loss of A1. Therefore, A1 appears to be a surprisingly redundant pro-survival protein in the haematopoietic system and other tissues, suggesting that its targeting in cancer may be readily tolerated.

Cognate antigen engagement on parenchymal cells stimulates CD8+ T cell proliferation in situ.

T-cell responses are initiated upon cognate presentation by professional antigen presenting cells in lymphoid tissue. T cells then migrate to inflamed tissues, but further T-cell stimulation in these parenchymal target sites is not well understood. Here we show that T-cell expansion within inflamed tissues is a distinct phase that is neither a classical primary nor classical secondary response. This response, which we term 'the mezzanine response', commences within days after initial antigen encounter, unlike the secondary response that usually occurs weeks after priming. A further distinction of this response is that T-cell proliferation is driven by parenchymal cell antigen presentation, without requiring professional antigen presenting cells, but with increased dependence on IL-2. The mezzanine response might, therefore, be a new target for inhibiting T-cell responses in allograft rejection and autoimmunity or for enhancing T-cell responses in the context of microbial or tumour immunity.

Stochastic Measurement Models for Quantifying Lymphocyte Responses Using Flow Cytometry.

Adaptive immune responses are complex dynamic processes whereby B and T cells undergo division and differentiation triggered by pathogenic stimuli. Deregulation of the response can lead to severe consequences for the host organism ranging from immune deficiencies to autoimmunity. Tracking cell division and differentiation by flow cytometry using fluorescent probes is a major method for measuring progression of lymphocyte responses, both in vitro and in vivo. In turn, mathematical modeling of cell numbers derived from such measurements has led to significant biological discoveries, and plays an increasingly important role in lymphocyte research. Fitting an appropriate parameterized model to such data is the goal of these studies but significant challenges are presented by the variability in measurements. This variation results from the sum of experimental noise and intrinsic probabilistic differences in cells and is difficult to characterize analytically. Current model fitting methods adopt different simplifying assumptions to describe the distribution of such measurements and these assumptions have not been tested directly. To help inform the choice and application of appropriate methods of model fitting to such data we studied the errors associated with flow cytometry measurements from a wide variety of experiments. We found that the mean and variance of the noise were related by a power law with an exponent between 1.3 and 1.8 for different datasets. This violated the assumptions inherent to commonly used least squares, linear variance scaling and log-transformation based methods. As a result of these findings we propose a new measurement model that we justify both theoretically, from the maximum entropy standpoint, and empirically using collected data. Our evaluation suggests that the new model can be reliably used for model fitting across a variety of conditions. Our work provides a foundation for modeling measurements in flow cytometry experiments thus facilitating progress in quantitative studies of lymphocyte responses.

Immunogenicity and safety of Advax™, a novel polysaccharide adjuvant based on delta inulin, when formulated with hepatitis B surface antigen: a randomized controlled Phase 1 study.

There is a need for additional safe and effective human vaccine adjuvants. Advax™ is a novel adjuvant produced from semi-crystalline particles of delta inulin. In animal studies Advax enhanced humoral and cellular immunity to hepatitis B surface antigen (HBsAg) without inducing local or systemic reactogenicity. This first-in-man Phase 1 clinical trial tested the safety and tolerability of three intramuscular doses of HBsAg formulated with Advax in a group of healthy adult subjects. Advax was well tolerated with injection site pain scores not significantly different to subjects receiving HBsAg alone and no adverse events were reported in subjects that received Advax. Seroprotection and HBsAb geometric mean titers (GMT) after three immunizations were higher in the Advax 5mg (seroprotection 5/6, 83.3%, GMT 40.7, 95% CI 11.9-139.1) and 10mg (seroprotection 4/5, 80%, GMT 51.6, 95% CI 10.0-266.2) groups versus HBsAg alone (seroprotection 1/5, 20%, GMT 4.1, 95% CI 1.3-12.8). Similarly the proportion of subjects with positive CD4 T-cell responses to HBsAg was higher in the Advax 5mg (4/6, 67%) and Advax 10mg (4/5, 80%) groups versus HBsAg alone (1/5, 20%). These results confirm the safety, tolerability and immunogenicity of Advax adjuvant observed in preclinical studies. Advax may represent a suitable replacement for alum adjuvants in prophylactic human vaccines subject to confirmation of current results in larger studies. Australia and New Zealand Clinical Trial Registry: ACTRN12607000598482.