Investigation of T cell signalling events regulating immunity and tolerance in vivo

Summary The ability of the immune system to discriminate between pathogenic and self or food antigens is essential not only for the generation of a productive immune response against invasive pathogens, but also for a state of antigen-specific tolerance to be elicited against harmless antigens. A breakdown in such tolerance can result in the development of a variety of autoimmune diseases including rheumatoid arthritis, Type 1 diabetes, inflammatory bowel disease and coeliac disease. Despite a wealth of studies in this field, however, the mechanisms by which the immune system can distinguish harmless and pathogenic antigens remain to be fully elucidated. If these mechanisms were better understood, such information could be exploited to help develop better therapies for autoimmune diseases, improve the rate of successful transplantations and increase the efficacy of vaccines. The primary means of maintaining tolerance to self antigens is to prevent self-reactive T cells from exiting the thymus following their development therein (central tolerance). However, some self-reactive T cells escape thymic deletion and as such, central tolerance is incomplete. Indeed, peripheral tolerance is required for an individual to elicit tolerance to all self-antigens, developmental antigens and some food and environmental antigens which are not present in early life. Peripheral tolerance is defined as a state of antigen-specific hyporesponsiveness, which is induced by exposure of T cells to antigen under sub-optimal activating conditions. For a T cell to become fully activated, and therefore productively primed, it must recognise its cognate antigen in the context of MHC and receive co-stimulation via the interaction of its CD28 receptor with CD80/86 on an antigen-presenting cell (APC). Clonal anergy, one of the proposed mechanisms of peripheral tolerance, describes a state of long lasting unresponsiveness to antigen in the T cell. Such anergy is induced when the TcR is ligated in the absence of co-stimulation and can be evidenced, upon re-stimulation with antigen, by reduced IL-2 production, cell cycle progression and proliferation, relative to that observed in primed cells. It has been widely proposed that both qualitative and quantitative differences in T cell signalling may underlie the differential functional outcomes of priming and tolerance. However, the majority of these studies have relied upon biochemical assessment of signalling in T cell lines or clones, at the population level following polyclonal stimulation in vitro, and thus has led to the generation of conflicting data. Moreover, and most importantly, these data do not necessarily reflect the responses of individual antigen-specific T cells within their environmental niche within primary or secondary lymphoid tissue. In addition, as such data represents the responses of all cell types in the sample population at any one time, they do not provide any information pertaining to the differential kinetics, amplitude or subcellular localisation of signals generated by functionally distinct subgroups within the population. A relatively new technology, laser scanning cytometry (LSC), offers an attractive means of investigating such responses, as it essentially marries the quantitative capabilities of flow cytometric analysis of cells in suspension with the ability to analyse spatially the fluorescence of large numbers of individual cells, either in suspension or in tissue in a slide-based format. Moreover, the adoptive transfer system, in which limited numbers of TcR transgenic (Tg) T cells are distributed evenly throughout the thymus-dependent area of the lymph node, provides an attractive means of studying antigen-specific responses occurring at near physiological frequencies in situ. Such antigen-specific T cells can be readily distinguished from endogenous T cells by LSC, following fluorescent staining of their TcR, as they are sparsely situated amongst the endogenous T cell population within the lymph node. Use of the adoptive transfer system, in combination with LSC analysis, has therefore enabled the development, in this thesis, of a quantitative imaging technology with which to study T cell signalling in individual antigen-specific T cells in vitro and in situ. In T cells, the maintenance phase of anergy has been reported to reflect defective activation of transcription factors, such as c-Jun/c-Fos, that are involved in formation of the AP-1 complex, which is required for inducing transcription of the IL-2 gene and optimal activation and effector function of T cells. In turn, this appears to be determined by the lack of recruitment of the ERK, JNK and p38 MAPK signalling cascades. The small GTPase, Rap1, has long been implicated in such desensitisation of ERK, and the consequent reduced IL-2 production, observed in tolerised T cells. However, as noted above, the majority of these findings were obtained from in vitro studies of T cell lines or clones and as such are not necessarily representative of physiological responses of primary antigen-specific T cells in situ. This study therefore describes, for the first time, an inverse relationship between ERK activation (pERK) and Rap1 expression in individual primary antigen-specific T cells during the maintenance phases of tolerance and priming, both in vitro and in vivo. Analysis at the single cell level further revealed that the proportion of antigen-stimulated cells expressing pERK was lower in the anergic relative to primed groups in vitro and in vivo, and the few anergised T cells expressing pERK did so at a lower level than the primed cells in vitro. By contrast, Rap1 was found to be expressed in a greater proportion of anergic antigen-specific T cells, and at considerably higher levels, compared with primed T cells following re-stimulation with antigen both in vitro and in vivo. An additional inverse relationship was observed between pERK and Rap1, concerning their subcellular localisation, with pERK appearing to co-localise with lipid raft structures in primed but not anergic cells and Rap1 appearing to be targeted to lipid rafts in anergic but not primed cells. These data suggests that Rap1 may be up-regulated and recruited to the immunological synapse upon re-stimulation with Ag in anergic T cells and that such Rap1 localisation and expression may contribute to the downregulation of ERK recruitment and activation in these cells. It is important to note that this inverse relationship between the accumulation of Rap1 and antagonism of ERK activation was only observed during the maintenance, and not induction, phases of both systemic and oral tolerance in vivo. Furthermore, assessment of the activation status of downstream cell cycle modulators in priming and tolerance, revealed that downregulation of ERK activation and upregulation of p27kip1 might not be sufficient for maintenance of the anergic state, as indicated by G1 arrest, and hence, one or more additional negative signals may be required. Indeed, this study suggests that perhaps such a negative signal could be provided by the downregulation of p-Rb and/or increased expression of inactive cyclin dependent kinases (CDKs). In summary, defective ERK signalling correlates with the up-regulation of Rap1 expression in tolerised relative to primed antigen-specific CD4+ T cells during the maintenance phases of tolerance in vitro and in vivo. As this association occurs after the induction of both systemic and oral routes of tolerance, these data suggest that Rap1 antagonism of pERK signalling may play an important and general role in the maintenance of antigen-specific CD4+ T cell tolerance. Moreover, as oral tolerance induction has been proposed as a potential therapy for autoimmune disorders and, oral administration of compounds offers a more attractive route for drug delivery in humans, these findings may have potential clinical applications. By advancing our knowledge of these key signals in regulating tolerance and priming at the single cell level in vitro and in vivo, we will therefore increase our understanding of an important physiological process at the molecular level, ultimately leading to identification of potential targets for enhancing or inhibiting immunity and tolerance

Finishing the euchromatic sequence of the human genome

The sequence of the human genome encodes the genetic instructions for human physiology, as well as rich information about human evolution. In 2001, the International Human Genome Sequencing Consortium reported a draft sequence of the euchromatic portion of the human genome. Since then, the international collaboration has worked to convert this draft into a genome sequence with high accuracy and nearly complete coverage. Here, we report the result of this finishing process. The current genome sequence (Build 35) contains 2.85 billion nucleotides interrupted by only 341 gaps. It covers ∼99% of the euchromatic genome and is accurate to an error rate of ∼1 event per 100,000 bases. Many of the remaining euchromatic gaps are associated with segmental duplications and will require focused work with new methods. The near-complete sequence, the first for a vertebrate, greatly improves the precision of biological analyses of the human genome including studies of gene number, birth and death. Notably, the human enome seems to encode only 20,000-25,000 protein-coding genes. The genome sequence reported here should serve as a firm foundation for biomedical research in the decades ahead

Mortality and pulmonary complications in patients undergoing surgery with perioperative SARS-CoV-2 infection: an international cohort study

Background: The impact of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) on postoperative recovery needs to be understood to inform clinical decision making during and after the COVID-19 pandemic. This study reports 30-day mortality and pulmonary complication rates in patients with perioperative SARS-CoV-2 infection. Methods: This international, multicentre, cohort study at 235 hospitals in 24 countries included all patients undergoing surgery who had SARS-CoV-2 infection confirmed within 7 days before or 30 days after surgery. The primary outcome measure was 30-day postoperative mortality and was assessed in all enrolled patients. The main secondary outcome measure was pulmonary complications, defined as pneumonia, acute respiratory distress syndrome, or unexpected postoperative ventilation. Findings: This analysis includes 1128 patients who had surgery between Jan 1 and March 31, 2020, of whom 835 (74·0%) had emergency surgery and 280 (24·8%) had elective surgery. SARS-CoV-2 infection was confirmed preoperatively in 294 (26·1%) patients. 30-day mortality was 23·8% (268 of 1128). Pulmonary complications occurred in 577 (51·2%) of 1128 patients; 30-day mortality in these patients was 38·0% (219 of 577), accounting for 81·7% (219 of 268) of all deaths. In adjusted analyses, 30-day mortality was associated with male sex (odds ratio 1·75 [95% CI 1·28–2·40], p\textless0·0001), age 70 years or older versus younger than 70 years (2·30 [1·65–3·22], p\textless0·0001), American Society of Anesthesiologists grades 3–5 versus grades 1–2 (2·35 [1·57–3·53], p\textless0·0001), malignant versus benign or obstetric diagnosis (1·55 [1·01–2·39], p=0·046), emergency versus elective surgery (1·67 [1·06–2·63], p=0·026), and major versus minor surgery (1·52 [1·01–2·31], p=0·047). Interpretation: Postoperative pulmonary complications occur in half of patients with perioperative SARS-CoV-2 infection and are associated with high mortality. Thresholds for surgery during the COVID-19 pandemic should be higher than during normal practice, particularly in men aged 70 years and older. Consideration should be given for postponing non-urgent procedures and promoting non-operative treatment to delay or avoid the need for surgery. Funding: National Institute for Health Research (NIHR), Association of Coloproctology of Great Britain and Ireland, Bowel and Cancer Research, Bowel Disease Research Foundation, Association of Upper Gastrointestinal Surgeons, British Association of Surgical Oncology, British Gynaecological Cancer Society, European Society of Coloproctology, NIHR Academy, Sarcoma UK, Vascular Society for Great Britain and Ireland, and Yorkshire Cancer Research

MUSiC : a model-unspecific search for new physics in proton-proton collisions at root s=13TeV

Results of the Model Unspecific Search in CMS (MUSiC), using proton-proton collision data recorded at the LHC at a centre-of-mass energy of 13 TeV, corresponding to an integrated luminosity of 35.9 fb(-1), are presented. The MUSiC analysis searches for anomalies that could be signatures of physics beyond the standard model. The analysis is based on the comparison of observed data with the standard model prediction, as determined from simulation, in several hundred final states and multiple kinematic distributions. Events containing at least one electron or muon are classified based on their final state topology, and an automated search algorithm surveys the observed data for deviations from the prediction. The sensitivity of the search is validated using multiple methods. No significant deviations from the predictions have been observed. For a wide range of final state topologies, agreement is found between the data and the standard model simulation. This analysis complements dedicated search analyses by significantly expanding the range of final states covered using a model independent approach with the largest data set to date to probe phase space regions beyond the reach of previous general searches.Peer reviewe

Measurement of prompt open-charm production cross sections in proton-proton collisions at root s=13 TeV

The production cross sections for prompt open-charm mesons in proton-proton collisions at a center-of-mass energy of 13TeV are reported. The measurement is performed using a data sample collected by the CMS experiment corresponding to an integrated luminosity of 29 nb(-1). The differential production cross sections of the D*(+/-), D-+/-, and D-0 ((D) over bar (0)) mesons are presented in ranges of transverse momentum and pseudorapidity 4 < p(T) < 100 GeV and vertical bar eta vertical bar < 2.1, respectively. The results are compared to several theoretical calculations and to previous measurements.Peer reviewe

Search for new particles in events with energetic jets and large missing transverse momentum in proton-proton collisions at root s=13 TeV

A search is presented for new particles produced at the LHC in proton-proton collisions at root s = 13 TeV, using events with energetic jets and large missing transverse momentum. The analysis is based on a data sample corresponding to an integrated luminosity of 101 fb(-1), collected in 2017-2018 with the CMS detector. Machine learning techniques are used to define separate categories for events with narrow jets from initial-state radiation and events with large-radius jets consistent with a hadronic decay of a W or Z boson. A statistical combination is made with an earlier search based on a data sample of 36 fb(-1), collected in 2016. No significant excess of events is observed with respect to the standard model background expectation determined from control samples in data. The results are interpreted in terms of limits on the branching fraction of an invisible decay of the Higgs boson, as well as constraints on simplified models of dark matter, on first-generation scalar leptoquarks decaying to quarks and neutrinos, and on models with large extra dimensions. Several of the new limits, specifically for spin-1 dark matter mediators, pseudoscalar mediators, colored mediators, and leptoquarks, are the most restrictive to date.Peer reviewe

Observation of the Production of Three Massive Gauge Bosons at root s=13 TeV

The first observation is reported of the combined production of three massive gauge bosons (VVV with V = W, Z) in proton-proton collisions at a center-of-mass energy of 13 TeV. The analysis is based on a data sample recorded by the CMS experiment at the CERN LHC corresponding to an integrated luminosity of 137 fb(-1). The searches for individualWWW, WWZ, WZZ, and ZZZ production are performed in final states with three, four, five, and six leptons (electrons or muons), or with two same-sign leptons plus one or two jets. The observed (expected) significance of the combinedVVV production signal is 5.7 (5.9) standard deviations and the corresponding measured cross section relative to the standard model prediction is 1.02(-0.23)(+0.26). The significances of the individual WWW and WWZ production are 3.3 and 3.4 standard deviations, respectively. Measured production cross sections for the individual triboson processes are also reported

Measurement of the W gamma Production Cross Section in Proton-Proton Collisions at root s=13 TeV and Constraints on Effective Field Theory Coefficients

A fiducial cross section for W gamma production in proton-proton collisions is measured at a center-of-mass energy of 13 TeV in 137 fb(-1) of data collected using the CMS detector at the LHC. The W -> e nu and mu nu decay modes are used in a maximum-likelihood fit to the lepton-photon invariant mass distribution to extract the combined cross section. The measured cross section is compared with theoretical expectations at next-to-leading order in quantum chromodynamics. In addition, 95% confidence level intervals are reported for anomalous triple-gauge couplings within the framework of effective field theory.Peer reviewe

Reconstruction of signal amplitudes in the CMS electromagnetic calorimeter in the presence of overlapping proton-proton interactions

A template fitting technique for reconstructing the amplitude of signals produced by the lead tungstate crystals of the CMS electromagnetic calorimeter is described. This novel approach is designed to suppress the contribution to the signal of the increased number of out-of-time interactions per beam crossing following the reduction of the accelerator bunch spacing from 50 to 25 ns at the start of Run 2 of the LHC. Execution of the algorithm is sufficiently fast for it to be employed in the CMS high-level trigger. It is also used in the offline event reconstruction. Results obtained from simulations and from Run 2 collision data (2015-2018) demonstrate a substantial improvement in the energy resolution of the calorimeter over a range of energies extending from a few GeV to several tens of GeV.Peer reviewe

Development and validation of HERWIG 7 tunes from CMS underlying-event measurements

This paper presents new sets of parameters (“tunes”) for the underlying-event model of the HERWIG7 event generator. These parameters control the description of multiple-parton interactions (MPI) and colour reconnection in HERWIG7, and are obtained from a fit to minimum-bias data collected by the CMS experiment at s=0.9, 7, and 13Te. The tunes are based on the NNPDF 3.1 next-to-next-to-leading-order parton distribution function (PDF) set for the parton shower, and either a leading-order or next-to-next-to-leading-order PDF set for the simulation of MPI and the beam remnants. Predictions utilizing the tunes are produced for event shape observables in electron-positron collisions, and for minimum-bias, inclusive jet, top quark pair, and Z and W boson events in proton-proton collisions, and are compared with data. Each of the new tunes describes the data at a reasonable level, and the tunes using a leading-order PDF for the simulation of MPI provide the best description of the dat