High-affinity CD8 variants enhance the sensitivity of pMHCI antigen recognition via low-affinity TCRs.

CD8+ T cell-mediated recognition of peptide-major histocompatibility complex class I (pMHCI) molecules involves cooperative binding of the T cell receptor (TCR), which confers antigen specificity, and the CD8 coreceptor, which stabilizes the TCR/pMHCI complex. Earlier work has shown that the sensitivity of antigen recognition can be regulated in vitro by altering the strength of the pMHCI/CD8 interaction. Here, we characterized two CD8 variants with moderately enhanced affinities for pMHCI, aiming to boost antigen sensitivity without inducing non-specific activation. Expression of these CD8 variants in model systems preferentially enhanced pMHCI antigen recognition in the context of low-affinity TCRs. A similar effect was observed using primary CD4+ T cells transduced with cancer-targeting TCRs. The introduction of high-affinity CD8 variants also enhanced the functional sensitivity of primary CD8+ T cells expressing cancer-targeting TCRs, but comparable results were obtained using exogenous wild-type CD8. Specificity was retained in every case, with no evidence of reactivity in the absence of cognate antigen. Collectively, these findings highlight a generically applicable mechanism to enhance the sensitivity of low-affinity pMHCI antigen recognition, which could augment the therapeutic efficacy of clinically relevant TCRs.

CD8 coreceptor-mediated focusing can reorder the agonist hierarchy of peptide ligands recognized via the T cell receptor.

CD8+ T cells are inherently cross-reactive and recognize numerous peptide antigens in the context of a given major histocompatibility complex class I (MHCI) molecule via the clonotypically expressed T cell receptor (TCR). The lineally expressed coreceptor CD8 interacts coordinately with MHCI at a distinct and largely invariant site to slow the TCR/peptide-MHCI (pMHCI) dissociation rate and enhance antigen sensitivity. However, this biological effect is not necessarily uniform, and theoretical models suggest that antigen sensitivity can be modulated in a differential manner by CD8. We used two intrinsically controlled systems to determine how the relationship between the TCR/pMHCI interaction and the pMHCI/CD8 interaction affects the functional sensitivity of antigen recognition. Our data show that modulation of the pMHCI/CD8 interaction can reorder the agonist hierarchy of peptide ligands across a spectrum of affinities for the TCR.

Identification of uterine pacemaker regions at the myometrial-placental interface in the rat.

KEY POINTS: Coordinated contraction of the uterine smooth muscle is essential to parturition. Histologically and physiologically defined pacemaker structures have not been identified in uterine smooth muscle. Here we report combined electrophysiological and histological evidence of zones associated with pacemaker activity in the rat myometrium. Our method relies crucially on the integration of histological and electrophysiological data in an in silico three-dimensional reconstruction of the rat myometrium at 10 µm resolution. We find that myometrial/placental pacemaking zones are closely related with placental sites and the area of disruptive myometrial remodelling surrounding such sites. If analogues of the myometrial/placental pacemaking zone are present in the human, defining their histology and physiology will be important steps towards treatment of pre-term birth, pre-eclampsia, and postpartum haemorrhage. ABSTRACT: Coordinated uterine contractions are essential for delivering viable offspring in mammals. In contrast to other visceral smooth muscles, it is not known where excitation within the uterus is initiated, and no defined pacemaking region has hitherto been identified. Using multi-electrode array recordings and high-resolution computational reconstruction of the three-dimensional micro-structure of late pregnant rat uterus, we demonstrate that electrical potentials are initiated in distinct structures within the placental bed of individual implantation sites. These previously unidentified structures represent modified smooth muscle bundles that are derived from bridges between the longitudinal and circular layers. Coordinated implantation and encapsulation by invading trophoblast give rise to isolated placental/myometrial interface bundles that directly connect to the overlying longitudinal smooth muscle layer. Taken together, these observations imply that the anatomical structure of the uterus, combined with site-specific implantation, gives rise to emergent patterns of electrical activity that drive effective contractility during parturition.

Structural Mechanism Underpinning Cross-reactivity of a CD8+ T-cell Clone That Recognizes a Peptide Derived from Human Telomerase Reverse Transcriptase.

T-cell cross-reactivity is essential for effective immune surveillance but has also been implicated as a pathway to autoimmunity. Previous studies have demonstrated that T-cell receptors (TCRs) that focus on a minimal motif within the peptide are able to facilitate a high level of T-cell cross-reactivity. However, the structural database shows that most TCRs exhibit less focused antigen binding involving contact with more peptide residues. To further explore the structural features that allow the clonally expressed TCR to functionally engage with multiple peptide-major histocompatibility complexes (pMHCs), we examined the ILA1 CD8+ T-cell clone that responds to a peptide sequence derived from human telomerase reverse transcriptase. The ILA1 TCR contacted its pMHC with a broad peptide binding footprint encompassing spatially distant peptide residues. Despite the lack of focused TCR-peptide binding, the ILA1 T-cell clone was still cross-reactive. Overall, the TCR-peptide contacts apparent in the structure correlated well with the level of degeneracy at different peptide positions. Thus, the ILA1 TCR was less tolerant of changes at peptide residues that were at, or adjacent to, key contact sites. This study provides new insights into the molecular mechanisms that control T-cell cross-reactivity with important implications for pathogen surveillance, autoimmunity, and transplant rejection.

Darwin endures, despite disparagement.

Evolution lies at the heart of the life sciences, and Charles Darwin is a towering historical figure within evolutionary science. One testimony to his lasting influence is that declaring Darwin to have been wrong all along remains a provocative way to command attention. The present paper discusses various strands of 'Darwin was wrong' partisans and their divergent views and motives: some are looking to Darwin to justify or condemn the political ideologies that they support or reject; others are concerned with the corrupting influence that the bleak cosmic outlook of evolution is deemed to exert on the moral or religious rectitude of impressionable minds, or regard Darwinism as a direct assault on religion; philosophers question the very coherence of the entire enterprise; and certain biologists aspire to go down in history as even greater than Darwin. It is sobering to reflect that this diverse group is united only by their poor grasp of Darwin's theory of natural selection.

Occam's razor: from Ockham's via moderna to modern data science.

The principle of parsimony, also known as 'Occam's razor', is a heuristic dictum that is thoroughly familiar to virtually all practitioners of science: Aristotle, Newton, and many others have enunciated it in some form or other. Even though the principle is not difficult to comprehend as a general heuristic guideline, it has proved surprisingly resistant to being put on a rigorous footing - a difficulty that has become more pressing and topical with the 'big data' explosion. We review the significance of Occam's razor in the philosophical and theological writings of William of Ockham, and survey modern developments of parsimony in data science.

CD8+ T-cell specificity is compromised at a defined MHCI/CD8 affinity threshold.

The CD8 co-receptor engages peptide-major histocompatibility complex class I (pMHCI) molecules at a largely invariant site distinct from the T-cell receptor (TCR)-binding platform and enhances the sensitivity of antigen-driven activation to promote effective CD8+ T-cell immunity. A small increase in the strength of the pMHCI/CD8 interaction (~1.5-fold) can disproportionately amplify this effect, boosting antigen sensitivity by up to two orders of magnitude. However, recognition specificity is lost altogether with more substantial increases in pMHCI/CD8 affinity (~10-fold). In this study, we used a panel of MHCI mutants with altered CD8-binding properties to show that TCR-mediated antigen specificity is delimited by a pMHCI/CD8 affinity threshold. Our findings suggest that CD8 can be engineered within certain biophysical parameters to enhance the therapeutic efficacy of adoptive T-cell transfer irrespective of antigen specificity.

Optimal management of nutrient reserves in microorganisms under time-varying environmental conditions.

Intracellular reserves are a conspicuous feature of many bacteria; such internal stores are often present in the form of inclusions in which polymeric storage compounds are accumulated. Such reserves tend to increase in times of plenty and be used up in times of scarcity. Mathematical models that describe the dynamical nature of reserve build-up and use are known as "cell quota," "dynamic energy/nutrient budget," or "variable-internal-stores" models. Here we present a stoichiometrically consistent macro-chemical model that accounts for variable stores as well as adaptive allocation of building blocks to various types of catalytic machinery. The model posits feedback loops linking expression of assimilatory machinery to reserve density. The precise form of the "regulatory law" at the heart of such a loop expresses how the cell manages internal stores. We demonstrate how this "regulatory law" can be recovered from experimental data using several empirical data sets. We find that stores should be expected to be negligibly small in stable growth-sustaining environments, but prominent in environments characterised by marked fluctuations on time scales commensurate with the inherent dynamic time scale of the organismal system.

Reconstruction of Cell Surface Densities of Ion Pumps, Exchangers, and Channels from mRNA Expression, Conductance Kinetics, Whole-Cell Calcium, and Current-Clamp Voltage Recordings, with an Application to Human Uterine Smooth Muscle Cells.

Uterine smooth muscle cells remain quiescent throughout most of gestation, only generating spontaneous action potentials immediately prior to, and during, labor. This study presents a method that combines transcriptomics with biophysical recordings to characterise the conductance repertoire of these cells, the 'conductance repertoire' being the total complement of ion channels and transporters expressed by an electrically active cell. Transcriptomic analysis provides a set of potential electrogenic entities, of which the conductance repertoire is a subset. Each entity within the conductance repertoire was modeled independently and its gating parameter values were fixed using the available biophysical data. The only remaining free parameters were the surface densities for each entity. We characterise the space of combinations of surface densities (density vectors) consistent with experimentally observed membrane potential and calcium waveforms. This yields insights on the functional redundancy of the system as well as its behavioral versatility. Our approach couples high-throughput transcriptomic data with physiological behaviors in health and disease, and provides a formal method to link genotype to phenotype in excitable systems. We accurately predict current densities and chart functional redundancy. For example, we find that to evoke the observed voltage waveform, the BK channel is functionally redundant whereas hERG is essential. Furthermore, our analysis suggests that activation of calcium-activated chloride conductances by intracellular calcium release is the key factor underlying spontaneous depolarisations.

Inceptions of biomathematics from Lotka to Thom.

Mathematical biology occupies a special place at the interface between the physical, mathematical and life sciences. Is this interface merely a meeting point for dabblers venturing out of their own proper domains to work on problems of mutual interest? Or is it an incipient science in its own right, with its own particular character, principles, and practices? The past century has seen vast advances in the application of mathematical and physical ideas and techniques to biological problems, in the process transforming many of them almost beyond recognition. Nonetheless, the question of a biomathematics as a new kind of science remains open, despite several fascinating, if sometimes problematic, attempts.

Mathematical models of microbial growth and metabolism: a whole-organism perspective.

We review the principles underpinning the development of mathematical models of the metabolic activities of micro-organisms. Such models are important to understand and chart the substantial contributions made by micro-organisms to geochemical cycles, and also to optimise the performance of bioreactors that exploit the biochemical capabilities of these organisms. We advocate an approach based on the principle of dynamic allocation. We survey the biological background that motivates this approach, including nutrient assimilation, the regulation of gene expression, and the principles of microbial growth. In addition, we discuss the classic models of microbial growth as well as contemporary approaches. The dynamic allocation theory generalises these classic models in a natural manner and is readily amenable to the additional information provided by transcriptomics and proteomics approaches. Finally, we touch upon these organising principles in the context of the transition from the free-living unicellular mode of life to multicellularity.

Variable-Internal-Stores models of microbial growth and metabolism with dynamic allocation of cellular resources.

Variable-Internal-Stores models of microbial metabolism and growth have proven to be invaluable in accounting for changes in cellular composition as microbial cells adapt to varying conditions of nutrient availability. Here, such a model is extended with explicit allocation of molecular building blocks among various types of catalytic machinery. Such an extension allows a reconstruction of the regulatory rules employed by the cell as it adapts its physiology to changing environmental conditions. Moreover, the extension proposed here creates a link between classic models of microbial growth and analyses based on detailed transcriptomics and proteomics data sets. We ascertain the compatibility between the extended Variable-Internal-Stores model and the classic models, demonstrate its behaviour by means of simulations, and provide a detailed treatment of the uniqueness and the stability of its equilibrium point as a function of the availabilities of the various nutrients.

Identification of human viral protein-derived ligands recognized by individual MHCI-restricted T-cell receptors.

Evidence indicates that autoimmunity can be triggered by virus-specific CD8(+) T cells that crossreact with self-derived peptide epitopes presented on the cell surface by major histocompatibility complex class I (MHCI) molecules. Identification of the associated viral pathogens is challenging because individual T-cell receptors can potentially recognize up to a million different peptides. Here, we generate peptide length-matched combinatorial peptide library (CPL) scan data for a panel of virus-specific CD8(+) T-cell clones spanning different restriction elements and a range of epitope lengths. CPL scan data drove a protein database search limited to viruses that infect humans. Peptide sequences were ranked in order of likelihood of recognition. For all anti-viral CD8(+) T-cell clones examined in this study, the index peptide was either the top-ranked sequence or ranked as one of the most likely sequences to be recognized. Thus, we demonstrate that anti-viral CD8(+) T-cell clones are highly focused on their index peptide sequence and that 'CPL-driven database searching' can be used to identify the inciting virus-derived epitope for a given CD8(+) T-cell clone. Moreover, to augment access to CPL-driven database searching, we have created a publicly accessible webtool. Application of these methodologies in the clinical setting may clarify the role of viral pathogens in the etiology of autoimmune diseases.

Epitope specificity delimits the functional capabilities of vaccine-induced CD8 T cell populations.

Despite progress toward understanding the correlates of protective T cell immunity in HIV infection, the optimal approach to Ag delivery by vaccination remains uncertain. We characterized two immunodominant CD8 T cell populations generated in response to immunization of BALB/c mice with a replication-deficient adenovirus serotype 5 vector expressing the HIV-derived Gag and Pol proteins at equivalent levels. The Gag-AI9/H-2K(d) epitope elicited high-avidity CD8 T cell populations with architecturally diverse clonotypic repertoires that displayed potent lytic activity in vivo. In contrast, the Pol-LI9/H-2D(d) epitope elicited motif-constrained CD8 T cell repertoires that displayed lower levels of physical avidity and lytic activity despite equivalent measures of overall clonality. Although low-dose vaccination enhanced the functional profiles of both epitope-specific CD8 T cell populations, greater polyfunctionality was apparent within the Pol-LI9/H-2D(d) specificity. Higher proportions of central memory-like cells were present after low-dose vaccination and at later time points. However, there were no noteworthy phenotypic differences between epitope-specific CD8 T cell populations across vaccine doses or time points. Collectively, these data indicate that the functional and phenotypic properties of vaccine-induced CD8 T cell populations are sensitive to dose manipulation, yet constrained by epitope specificity in a clonotype-dependent manner.

X makes nine: a distant ice giant in the solar system.

Ever since Pluto lost its status as one of the main planets of our solar system and was demoted to just another frozen denizen of the Kuiper belt, we have had to make do with eight, albeit in a pleasing symmetry, with four rocky ones this side of the asteroid belt and four giants on the far side. Now it looks like number nine is back on the slate: the existence of a large planet, about ten times as massive as Earth and hundreds of times more distant from the Sun than Earth itself, has been postulated to explain the curiously bunched-up orbits of several small celestial bodies, far beyond the orbit of Neptune. To date, we have only "proof by simulation" and we are yet to observe this massive planet in the backyard of our solar system by more direct means. However, powerful new telescopes should provide visual evidence within the next few decades.

Beyond the Discovery Void: New targets for antibacterial compounds.

Antibiotics save many lives, but their efficacy is under threat: overprescription, population growth, and global travel all contribute to the rapid origination and spread of resistant strains. Exacerbating this threat is the fact that no new major classes of antibiotics have been discovered in the last 30 years: this is the "discovery void." We discuss the traditional molecular targets of antibiotics as well as putative novel targets.

Biological Insights from a Simulation Model of the Critical FtsZ Accumulation Required for Prokaryotic Cell Division.

A simulation model of prokaryotic Z-ring assembly, based on the observed behavior of FtsZ in vitro as well as on in vivo parameters, is used to integrate critical processes in cell division. According to the model, the cell's ability to divide depends on a "contraction parameter" (χ) that links the force of contraction to the dynamics of FtsZ. This parameter accurately predicts the outcome of division. Evaluating the GTP binding strength, the FtsZ polymerization rate, and the intrinsic GTP hydrolysis/dissociation activity, we find that inhibition of GTP-FtsZ binding is an inefficient antibacterial target. Furthermore, simulations indicate that the temperature sensitivity of the ftsZ84 mutation arises from the conversion of FtsZ to a dual-specificity NTPase. Finally, the sensitivity to temperature of the rate of ATP hydrolysis, over the critical temperature range, leads us to conclude that the ftsZ84 mutation affects the turnover rate of the Z-ring much less strongly than previously reported.

Peptide length determines the outcome of TCR/peptide-MHCI engagement.

αß-TCRs expressed at the CD8(+) T-cell surface interact with short peptide fragments (p) bound to MHC class I molecules (pMHCI). The TCR/pMHCI interaction is pivotal in all aspects of CD8(+) T-cell immunity. However, the rules that govern the outcome of TCR/pMHCI engagement are not entirely understood, and this is a major barrier to understanding the requirements for both effective immunity and vaccination. In the present study, we discovered an unexpected feature of the TCR/pMHCI interaction by showing that any given TCR exhibits an explicit preference for a single MHCI-peptide length. Agonists of nonpreferred length were extremely rare, suboptimal, and often entirely distinct in sequence. Structural analysis indicated that alterations in peptide length have a major impact on antigenic complexity, to which individual TCRs are unable to adapt. This novel finding demonstrates that the outcome of TCR/pMHCI engagement is determined by peptide length in addition to the sequence identity of the MHCI-bound peptide. Accordingly, the effective recognition of pMHCI Ag, which is a prerequisite for successful CD8(+) T-cell immunity and protective vaccination, can only be achieved by length-matched Ag-specific CD8(+) T-cell clonotypes.

Functional and morphological development of the womb throughout life.

The uterus undergoes changes throughout a woman's life, beginning with her own embryonic development when she is still in the womb, commencing a monthly cycle at the onset of adulthood, and undergoing dramatic changes during pregnancy and parturition. The impact of preterm labour and other perinatal health problems is significant, both in human and financial terms; therefore the study of the physiological and regulatory changes which the uterus undergoes can be of enormous potential benefit. Here we briefly review the current state of knowledge, with an emphasis on the importance of changes in connectivity in the uterine smooth muscle cell network and on recent mathematical modelling work aimed at elucidating the role of spatial heterogeneity in this connected network.

Bacterial cell division: experimental and theoretical approaches to the divisome.

Cell division is a key event in the bacterial life cycle. It involves constriction at the midcell, so that one cell can give rise to two daughter cells. This constriction is mediated by a ring composed offibrous multimers of the protein FtsZ. However a host of additional factors is involved in the formation and dynamics of this "Z-ring" and this complicated apparatus is collectively known as the "divisome". We review the literature, with an emphasis on mathematical modelling, and show how such theoretical efforts have helped experimentalists to make sense of the at times bewildering data, and plan further experiments.