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RationalePersistent pulmonary sequelae are evident in many survivors of acute coronavirus disease 2019 (COVID-19) but the molecular mechanisms responsible are incompletely understood. Post-COVID radiological lung abnormalities comprise two broad categories, organising pneumonia and reticulation, interpreted as indicative of subacute inflammation and fibrosis, respectively. Whether these two patterns represent distinct pathologies, likely to require different treatment strategies is not known.ObjectivesWe sought to identify differences at molecular and cellular level, in the local immunopathology of post-COVID inflammation and fibrosis.MethodsWe compared single-cell transcriptomic profiles and T cell receptor (TCR) repertoires of bronchoalveolar cells obtained from convalescent individuals with each radiological pattern of post-COVID lung disease (PCLD).Measurements and Main ResultsInflammatory and fibrotic PCLD single-cell transcriptomes closely resembled each other across all cell types. However, CD4 central memory T cells (TCM) and CD8 effector memory T cells (TEM) were significantly more abundant in inflammatory PCLD. A greater proportion of CD4 TCM also exhibited clonal expansion in inflammatory PCLD. High levels of clustering of similar TCRs from multiple donors was a striking feature of both PCLD phenotypes, consistent with tissue localised antigen-specific immune responses, but there was no enrichment for known SARS-CoV-2 reactive TCRs.ConclusionsThere is no evidence that radiographic organising pneumonia and reticulation in PCLD are associated with differential immmunopathological pathways. Inflammatory radiology is characterised by greater bronchoalveolar T cell accumulation. Both groups show evidence of shared antigen-specific T cell responses, but the antigenic target for these T cells remains to be identified.Scientific knowledge on the subjectThe immune pathogenesis of persistent pulmonary radiological abnormalities following COVID-19 is poorly understood. Whether post-COVID radiological inflammation and fibrosis represent distinct disease entities with different molecular mechanisms of tissue injury is not known.What this study adds to the fieldSingle-cell bronchoalveolar transcriptomes of inflammatory and fibrotic post-COVID lung disease closely resemble each other across all cell types, but CD4 central memory and CD8 effector memory T cells are more abundant in the inflammatory group. Marked T cell receptor clustering, suggestive of antigen-specific responses is evident in both phenotypes. These two radiological patterns likely represent different manifestations of the same disease process, which may benefit from therapies which target T cells.

C-C chemokine receptor 5 (CCR5) is a major co-receptor molecule used by HIV-1 to enter cells. Two X-ray crystallographic studies are presented of the antibody RoAb13, which binds to the peptide PIYDIN, which is part of the N-terminal domain of CCR5. The results may provide the basis for active immunization vaccines to stimulate an antibody response to native CCR5 that will block HIV infection., C-C chemokine receptor 5 (CCR5) is a major co-receptor molecule used by HIV-1 to enter cells. This led to the hypothesis that stimulating an antibody response would block HIV with minimal toxicity. Here, X-ray crystallographic studies of the anti-CCR5 antibody RoAb13 together with two peptides were undertaken: one peptide is a 31-residue peptide containing the PIYDIN sequence and the other is the PIDYIN peptide alone, where PIYDIN is part of the N-terminal region of CCR5 previously shown to be important for HIV entry. In the presence of the longer peptide (the complete N-terminal domain), difference electron density was observed at a site within a hypervariable CDR3 binding region. In the presence of the shorter core peptide PIYDIN, difference electron density is again observed at this CDR3 site, confirming consistent binding for both peptides. This may be useful in the design of a new biomimetic to stimulate an antibody response to CCR5 in order to block HIV infection.

A boost from infection During clinical trials of severe acute respiratory syndrome coronavirus 2 vaccines, no one who had survived infection with the virus was tested. A year after the pandemic was declared, vaccination of previously infected persons is a reality. Reynolds et al. address the knowledge gap in a cohort of UK health care workers given the Pfizer/BioNTech vaccine in which half of the participants had experienced natural virus infections early in the pandemic (see the Perspective by Crotty). Genotyping indicated that a genetic component underlies heterogeneity in immune responses to vaccine and to natural infection. After vaccination, naïve individuals developed antibody responses similar to those seen in naturally infected persons, but T cell responses were more limited and sometimes absent. However, antibody and memory responses in individuals vaccinated after infection were substantially boosted to the extent that a single vaccine dose is likely to protect against the more aggressive B.1.1.7 variant. It is possible that the messenger RNA vaccine has an adjuvant effect, biasing responses toward antibody generation. Science , abh1282, this issue p. 1418 ; see also abj2258, p. 1392

Effective control of SARS-CoV-2 infection on primary exposure may reveal correlates of protective immunity to future variants, but we lack insights into immune responses before or at the time virus is first detected. We use blood transcriptomics, multiparameter flow cytometry, and T cell receptor (TCR) sequencing spanning the time of incident non-severe infection in unvaccinated virus-naive individuals to identify rapid type 1 interferon (IFN) responses common to other acute respiratory viruses and cell proliferation responses that discriminate SARS-CoV-2 from other viruses. These peak by the time the virus is first detected and sometimes precede virus detection. Cell proliferation is most evident in CD8 T cells and associated with specific expansion of SARS-CoV-2-reactive TCRs, in contrast to virus-specific antibodies, which lag by 1-2 weeks. Our data support a protective role for early type 1 IFN and CD8 T cell responses, with implications for development of universal T cell vaccines.

TCR-gene-transfer is an efficient strategy to produce therapeutic T cells of defined antigen specificity. However, there are substantial variations in the cell surface expression levels of human TCRs, which can impair the function of engineered T cells. Here we demonstrate that substitutions of 3 amino acid residues in the framework of the TCR variable domains consistently increase the expression of human TCRs on the surface of engineered T cells.The modified TCRs mediate enhanced T cell proliferation, cytokine production and cytotoxicity, while reducing the peptide concentration required for triggering effector function up to 3000-fold. Adoptive transfer experiments in mice show that modified TCRs control tumor growth more efficiently than wild-type TCRs. Our data indicate that simple variable domain modifications at a distance from the antigen-binding loops lead to increased TCR expression and improved effector function. This finding provides a generic platform to optimize the efficacy of TCR gene therapy in humans., Increasing TCR cell surface expression can potentiate T cell responses to low-concentrations of antigen. Here the authors identify aminoacids in human TCR variable domains that impact its surface expression, and demonstrate how editing these residues can improve T cell activation and effector function without altering antigen specificity.

The COVID-19 pandemic has pushed healthcare systems globally to a breaking point. The urgent need for effective and affordable COVID-19 treatments calls for repurposing combinations of approved drugs. The challenge is to identify which combinations are likely to be most effective and at what stages of the disease. Here, we present the first disease-stage executable signalling network model of SARS-CoV-2-host interactions used to predict effective repurposed drug combinations for treating early- and late-stage severe disease. Using our executable model, we performed in silico screening of 9870 pairs of 140 potential targets and have identified 12 new drug combinations. Camostat and Apilimod were predicted to be the most promising combination in effectively supressing viral replication in the early stages of severe disease and were validated experimentally in human Caco-2 cells. Our study further demonstrates the power of executable mechanistic modelling to enable rapid pre-clinical evaluation of combination therapies tailored to disease progression. It also presents a novel resource and expandable model system that can respond to further needs in the pandemic.

Summary Background We hypothesised that host-response biomarkers of viral infections might contribute to early identification of individuals infected with SARS-CoV-2, which is critical to breaking the chains of transmission. We aimed to evaluate the diagnostic accuracy of existing candidate whole-blood transcriptomic signatures for viral infection to predict positivity of nasopharyngeal SARS-CoV-2 PCR testing. Methods We did a nested case-control diagnostic accuracy study among a prospective cohort of health-care workers (aged ≥18 years) at St Bartholomew's Hospital (London, UK) undergoing weekly blood and nasopharyngeal swab sampling for whole-blood RNA sequencing and SARS-CoV-2 PCR testing, when fit to attend work. We identified candidate blood transcriptomic signatures for viral infection through a systematic literature search. We searched MEDLINE for articles published between database inception and Oct 12, 2020, using comprehensive MeSH and keyword terms for "viral infection", "transcriptome", "biomarker", and "blood". We reconstructed signature scores in blood RNA sequencing data and evaluated their diagnostic accuracy for contemporaneous SARS-CoV-2 infection, compared with the gold standard of SARS-CoV-2 PCR testing, by quantifying the area under the receiver operating characteristic curve (AUROC), sensitivities, and specificities at a standardised Z score of at least 2 based on the distribution of signature scores in test-negative controls. We used pairwise DeLong tests compared with the most discriminating signature to identify the subset of best performing biomarkers. We evaluated associations between signature expression, viral load (using PCR cycle thresholds), and symptom status visually and using Spearman rank correlation. The primary outcome was the AUROC for discriminating between samples from participants who tested negative throughout the study (test-negative controls) and samples from participants with PCR-confirmed SARS-CoV-2 infection (test-positive participants) during their first week of PCR positivity. Findings We identified 20 candidate blood transcriptomic signatures of viral infection from 18 studies and evaluated their accuracy among 169 blood RNA samples from 96 participants over 24 weeks. Participants were recruited between March 23 and March 31, 2020. 114 samples were from 41 participants with SARS-CoV-2 infection, and 55 samples were from 55 test-negative controls. The median age of participants was 36 years (IQR 27–47) and 69 (72%) of 96 were women. Signatures had little overlap of component genes, but were mostly correlated as components of type I interferon responses. A single blood transcript for IFI27 provided the highest accuracy for discriminating between test-negative controls and test-positive individuals at the time of their first positive SARS-CoV-2 PCR result, with AUROC of 0·95 (95% CI 0·91–0·99), sensitivity 0·84 (0·70–0·93), and specificity 0·95 (0·85–0·98) at a predefined threshold (Z score >2). The transcript performed equally well in individuals with and without symptoms. Three other candidate signatures (including two to 48 transcripts) had statistically equivalent discrimination to IFI27 (AUROCs 0·91–0·95). Interpretation Our findings support further urgent evaluation and development of blood IFI27 transcripts as a biomarker for early phase SARS-CoV-2 infection for screening individuals at high risk of infection, such as contacts of index cases, to facilitate early case isolation and early use of antiviral treatments as they emerge. Funding Barts Charity, Wellcome Trust, and National Institute of Health Research.

T cell receptor (TCR) sequencing has emerged as a powerful new technology in analysis of the host-tumour interaction. The advances in NextGen sequencing technologies, coupled with powerful novel bioinformatic tools, allow quantitative and reproducible characterisation of repertoires from tumour and blood samples from an increasing number of patients with a variety of solid cancers. In this review, we consider how global metrics such as T cell clonality and diversity can be extracted from these repertoires and used to give insight into the mechanism of action of immune checkpoint blockade. Furthermore, we explore how the analysis of TCR overlap between repertories can help define spatial and temporal heterogeneity of the anti-tumoural immune response. Finally, we review how analysis of TCR sequence and structure, either of individual TCRs or from sets of related TCRs can be used to annotate the antigenic specificity, with important implications for the development of personalised adoptive cellular immunotherapies.

The correlates of natural protective immunity to SARS-CoV-2 in the majority who experience asymptomatic infection or non-severe disease are not fully characterised, and remain important as new variants emerge. We addressed this question using blood transcriptomics, multiparameter flow cytometry and T cell receptor (TCR) sequencing spanning the time of incident infection. We identified a type 1 interferon (IFN) response common to other acute respiratory viruses, and a cell proliferation response that discriminated SARS-CoV-2 from other viruses. These responses peaked by the time the virus was first detected, and in some preceded virus detection. Cell proliferation was most evident in CD8 T cells and associated with rapid expansion of SARS-CoV-2 reactive TCRs. We found an equally rapid increase in immunoglobulin transcripts, but circulating virus-specific antibodies lagged by 1-2 weeks. Our data support a protective role for rapid induction of type 1 IFN and CD8 T cell responses to SARS-CoV-2.

We hypothesised that host-response biomarkers of viral infections may contribute to early identification of SARS-CoV-2 infected individuals, critical to breaking chains of transmission. We identified 20 candidate blood transcriptomic signatures of viral infection by systematic review and evaluated their ability to detect SARS-CoV-2 infection, compared to the gold-standard of virus PCR tests, among a prospective cohort of 400 hospital staff subjected to weekly testing when fit to attend work. The transcriptional signatures had limited overlap, but were mostly co-correlated as components of type 1 interferon responses. We reconstructed each signature score in blood RNA sequencing data from 41 individuals over sequential weeks spanning a first positive SARS-CoV-2 PCR, and after 6-month convalescence. A single blood transcript for IFI27 provided the highest accuracy for discriminating individuals at the time of their first positive viral PCR result from uninfected controls, with area under the receiver operating characteristic curve (AUROC) of 0.95 (95% confidence interval 0.91–0.99), sensitivity 0.84 (0.7–0.93) and specificity 0.95 (0.85–0.98) at a predefined test threshold. The test performed equally well in individuals with and without symptoms, correlated with viral load, and identified incident infections one week before the first positive viral PCR with sensitivity 0.4 (0.17–0.69) and specificity 0.95 (0.85–0.98). Our findings strongly support further urgent evaluation and development of blood IFI27 transcripts as a biomarker for early phase SARS-CoV-2 infection, for screening individuals such as contacts of index cases, in order to facilitate early case isolation and early antiviral treatments as they emerge.

BACKGROUND: SARS-CoV-2 serology is used to identify prior infection at individual and at population level. Extended longitudinal studies with multi-timepoint sampling to evaluate dynamic changes in antibody levels are required to identify the time horizon in which these applications of serology are valid, and to explore the longevity of protective humoral immunity. METHODS: Healthcare workers were recruited to a prospective cohort study from the first SARS-CoV-2 epidemic peak in London, undergoing weekly symptom screen, viral PCR and blood sampling over 16-21 weeks. Serological analysis (n =12,990) was performed using semi-quantitative Euroimmun IgG to viral spike S1 domain and Roche total antibody to viral nucleocapsid protein (NP) assays. Comparisons were made to pseudovirus neutralizing antibody measurements. FINDINGS: A total of 157/729 (21.5%) participants developed positive SARS-CoV-2 serology by one or other assay, of whom 31.0% were asymptomatic and there were no deaths. Peak Euroimmun anti-S1 and Roche anti-NP measurements correlated (r = 0.57, p

Understanding the nature of immunity following mild/asymptomatic infection with SARS-CoV-2 is crucial to controlling the pandemic. We analyzed T cell and neutralizing antibody responses in 136 healthcare workers (HCW) 16-18 weeks after United Kingdom lockdown, 76 of whom had mild/asymptomatic SARS-CoV-2 infection captured by serial sampling. Neutralizing antibodies (nAb) were present in 89% of previously infected HCW. T cell responses tended to be lower following asymptomatic infection than in those reporting case-definition symptoms of COVID-19, while nAb titers were maintained irrespective of symptoms. T cell and antibody responses were sometimes discordant. Eleven percent lacked nAb and had undetectable T cell responses to spike protein but had T cells reactive with other SARS-CoV-2 antigens. Our findings suggest that the majority of individuals with mild or asymptomatic SARS-CoV-2 infection carry nAb complemented by multispecific T cell responses at 16-18 weeks after mild or asymptomatic SARS-CoV-2 infection., Uncoupling of neutralizing antibody and T cell responses to SARS-CoV-2 antigens occurs in some mild or asymptomatic cases of COVID-19.

Studies of adaptive immunity to SARS-CoV-2 include characterisation of lethal, severe and mild cases1-8. Understanding how long immunity lasts in people who have had mild or asymptomatic infection is crucial. Healthcare worker (HCW) cohorts exposed to and infected by SARS-CoV-2 during the early stages of the pandemic are an invaluable resource to study this question9-14. The UK COVIDsortium is a longitudinal, London hospital HCW cohort, followed from the time of UK lockdown9,10 ; weekly PCR, serology and symptom diaries allowed capture of asymptomatic infection around the time of onset, so duration of immunity could be tracked. Here, we conduct a cross-sectional, case-control, sub-study of 136 HCW at 16-18 weeks after UK lockdown, with 76 having had laboratory-confirmed SARS-CoV-2 mild or asymptomatic infection. Neutralising antibodies (nAb) were present in 90% of infected HCW sampled after the first wave; titres, likely to correlate with functional protection, were present in 66% at 16-18 weeks. T cell responses tended to be lower in asymptomatic infected HCW than those reporting case-definition symptoms of COVID-19, while nAb titres were maintained irrespective of symptoms. T cell and antibody responses were discordant. HCW lacking nAb also showed undetectable T cells to Spike protein but had T cells of other specificities. Our findings suggest that the majority of HCW with mild or asymptomatic SARS-CoV-2 infection carry nAb complemented by multi-specific T cell responses for at least 4 months after mild or asymptomatic SARS-CoV-2 infection.

Background Effective anti-tumour immunity requires cancer antigen expression, but persistent antigen exposure in chronic viral infections and autoimmunity has a detrimental effect on immune function. This is associated with a decline of early differentiated T cell populations in favour of later differentiated, dysfunctional subsets, resulting in an unfavourable skewing of the immune landscape. It is unknown whether this occurs locally within the antigen rich tumour microenvironment, driving immune failure. Materials and Methods We combined tumour infiltrating lymphocyte (TIL) high dimensional flow cytometry, bulk exome and RNA sequencing data from multiregional samples obtained from surgically resected tumours of treatment naive patients with non-small cell lung cancer (NSCLC) amongst the first 100 recruited to the prospective, UK-wide lung TRACERx study. Clonal relationship between T cell populations was determined by T cell receptor (TCR) sequencing. We additionally analysed publically available single T cell RNA sequencing data and bulk RNA sequencing data within TCGA. Results T cell differentiation skewing (TDS) occurred amongst TILs in association with tumour mutational burden (TMB). Surprisingly, this was most evident within the CD4 compartment that had a greater abundance of central memory cells expressing the key transcription factor TCF7. Amongst CD4 cells, loss of a PD1-CCR7+ T central memory population was accompanied by gain in abundance of PD1+ populations with exhausted (CD57-ICOShiCTLA4hi) and terminally differentiated effector (CD57+Eomes+) features. TCR sequencing revealed early and dysfunctional differentiated populations to be clonally related and CDR3 clustering analysis showed greater similarity of sequences shared vs. non-shared between subsets, consistent with an antigen driven differentiation process. Similar patterns were observed within the CD8 compartment. Identification of these subsets within single T cell RNA sequencing data revealed shared and distinct functional regulators, suggesting the enhanced effector capability of early compared to dysfunctionally differentiated populations. A validated transcriptional signature of TDS generated using TRACERx samples with paired flow cytometry and RNA sequencing data reflected loss of gene expression downstream of TCF7, and predicted worse survival within TRACERx and multiple TCGA cohorts including lung adenocarcinoma (LUAD). Conclusions Our finding support a model of neoantigen driven T cell differentiation within the tumour microenvironment that drives the depletion of progenitor-like cells and gain in abundance of dysfunctional subsets, resulting in a loss of immune fitness. Our analysis of transcriptomic data elucidates potential regulatory mechanisms and therapeutic targets within the subsets identified. Disclosure Information E. Ghorani: None. J. Reading: None. J. Henry: None. M. Robert de Massy: None. R. Rosenthal: E. Ownership Interest (stock, stock options, patent or other intellectual property); Modest; Achilles Therapeutics. F. Consultant/Advisory Board; Modest; Achilles Therapeutics. V. Turati: None. A. Furness: None. A. Ben Aissa: None. S. Kumar Saini: None. S. Ramskov: None. A. Georgiou: None. M. Vila De Mucha: None. I. Uddin: None. T. Ronel: None. R. Salgado: None. T. Lund: None. J. Herrero: None. T. Enver: None. S. Hadrup: None. A. Hackshaw: None. K. Peggs: E. Ownership Interest (stock, stock options, patent or other intellectual property); Modest; Achilles Therapeutics. N. McGranahan,: E. Ownership Interest (stock, stock options, patent or other intellectual property); Modest; Achilles Therapeutics. F. Consultant/Advisory Board; Modest; Achilles Therapeutics. B. Chain: None. C. Swanton: B. Research Grant (principal investigator, collaborator or consultant and pending grants as well as grants already received); Modest; Pfizer, AstraZeneca, BMS, Roche–Ventana and Boehringer Ingelheim. E. Ownership Interest (stock, stock options, patent or other intellectual property); Modest; ApoGen Biotechnologies, Epic Bioscience and GRAIL, and has stock options in and is co-founder of Achilles Therapeutics. F. Consultant/Advisory Board; Modest; Pfizer, Novartis, GlaxoSmithKline, MSD, BMS, Celgene, AstraZeneca, Illumina, Genentech, Roche–Ventana, GRAIL, Medicxi and the Sarah Cannon Research Institute. S. Quezada: E. Ownership Interest (stock, stock options, patent or other intellectual property); Modest; Achilles Therapeutics.

Background There is an urgent need to develop biomarkers that stratify risk of bacterial infection in order to support antimicrobial stewardship in emergency hospital admissions. Methods We used computational machine learning to derive a rule-out blood transcriptomic signature of bacterial infection (SeptiCyte™ TRIAGE) from eight published case-control studies. We then validated this signature by itself in independent case-control data from more than 1500 samples in total, and in combination with our previously published signature for viral infections (SeptiCyte™ VIRUS) using pooled data from a further 1088 samples. Finally, we tested the performance of these signatures in a prospective observational cohort of emergency department (ED) patients with fever, and we used the combined SeptiCyte™ signature in a mixture modelling approach to estimate the prevalence of bacterial and viral infections in febrile ED patients without microbiological diagnoses. Results The combination of SeptiCyte™ TRIAGE with our published signature for viral infections (SeptiCyte™ VIRUS) discriminated bacterial and viral infections in febrile ED patients, with a receiver operating characteristic area under the curve of 0.95 (95% confidence interval 0.90–1), compared to 0.79 (0.68–0.91) for WCC and 0.73 (0.61–0.86) for CRP. At pre-test probabilities 0.35 and 0.72, the combined SeptiCyte™ score achieved a negative predictive value for bacterial infection of 0.97 (0.90–0.99) and 0.86 (0.64–0.96), compared to 0.90 (0.80–0.94) and 0.66 (0.48–0.79) for WCC and 0.88 (0.69–0.95) and 0.60 (0.31–0.72) for CRP. In a mixture modelling approach, the combined SeptiCyte™ score estimated that 24% of febrile ED cases receiving antibacterials without a microbiological diagnosis were due to viral infections. Our analysis also suggested that a proportion of patients with bacterial infection recovered without antibacterials. Conclusions Blood transcriptional biomarkers offer exciting opportunities to support precision antibacterial prescribing in ED and improve diagnostic classification of patients without microbiologically confirmed infections.

Background and aims Learning from error can have a negative impact on the staff involved in the error (‘second victim phenomenon’1). We created a project, based on the principles of the Learning from Excellence project,2 to learn from excellence and correct the imbalance of negative to positive feedback in the context of hospital practice. Methods and results Using a questionnaire, we surveyed staff on existing feedback mechanisms and morale. We then introduced a system where staff recorded and commented on examples of excellence in practice. Recipients and their supervisors received copies of these reports and the feedback was analysed and discussed with senior staff (consultant, senior charge nurse, managers). We re-audited the staff two months after starting this project and noted improvements in staff morale and in positive reporting. Conclusions This project has improved the process of giving and learning from positive feedback and had a significant impact on staff morale. We can also demonstrate an example of improved clinical practice (from feedback received) and will now attempt to measure clinical outcomes with a new prospective study. Finally, we hope to set up a regional programme of reporting excellence in South-East Scotland.

Background: Host immune responses at the site of Mycobacterium tuberculosis (Mtb) infection serve to contain the pathogen, but also mediate the pathogenesis of tuberculosis (TB) and onward transmission of infection. Based on the premise that active TB disease is predominantly a manifestation of immunopathology, we tested the hypothesis that immune responses at the site of host-pathogen interactions would reveal enrichment of immunopathologic responses in patients with active TB that were absent in individuals with equivalent immune memory for Mtb but without disease. Methods: In cohorts of patients with active TB and cured or latent infection, we undertook molecular profiling at the site of a tuberculin skin test to model in vivo host-pathogen interactions in Mtb infection. Genome-wide transcriptional differences were identified by differential gene expression analyses. Enrichment of immune cells and cytokine activity was derived using specific transcriptional modules. Findings were validated in independent cohorts of patients with active TB, as well as Mtb infected tissues. Results: Active TB in humans is associated with exaggerated IL-17A/F expression, accumulation of Th17 cells and IL-17A bioactivity, including increased neutrophil recruitment and matrix metalloproteinase-1 expression directly implicated in TB pathogenesis. These features discriminate recall responses in patients with active TB from those with cured or latent infection, and are also evident at the site of TB disease. Conclusions: Our data are consistent with a model in which elevated Th17 responses within tissues drive immunopathology and transmission in active TB, and support targeting of the IL-17A/F pathway in host-directed therapy for active TB.

The highest risk of tuberculosis arises in the first few months after exposure. We reasoned that this risk reflects incipient disease among tuberculosis contacts. Blood transcriptional biomarkers of tuberculosis may predate clinical diagnosis, suggesting they offer improved sensitivity to detect subclinical incipient disease. Therefore, we sought to test the hypothesis that refined blood transcriptional biomarkers of active tuberculosis will improve stratification of short-term disease risk in tuberculosis contacts.We combined analysis of previously published blood transcriptomic data with new data from a prospective human immunodeficiency virus (HIV)-negative UK cohort of 333 tuberculosis contacts. We used stability selection as an alternative computational approach to identify an optimal signature for short-term risk of active tuberculosis and evaluated its predictive value in independent cohorts.In a previously published HIV-negative South African case-control study of patients with asymptomatic Mycobacterium tuberculosis infection, a novel 3-gene transcriptional signature comprising BATF2, GBP5, and SCARF1 achieved a positive predictive value (PPV) of 23% for progression to active tuberculosis within 90 days. In a new UK cohort of 333 HIV-negative tuberculosis contacts with a median follow-up of 346 days, this signature achieved a PPV of 50% (95% confidence interval [CI], 15.7-84.3) and negative predictive value of 99.3% (95% CI, 97.5-99.9). By comparison, peripheral blood interferon gamma release assays in the same cohort achieved a PPV of 5.6% (95% CI, 2.1-11.8).This blood transcriptional signature provides unprecedented opportunities to target therapy among tuberculosis contacts with greatest risk of incident disease.

Objective Pseudomonas aeruginosa effectively facilitate resistance to phagocyte killing by biofilm formation. However,b the cross talk between biofilm components and phagocytes is still unclear. We hypothesize that a biofilm provides a concentrated extracellular source of LPS, DNA and exopolysaccharides (EPS), which polarize neighbouring phagocytes into an adverse hyperinflammatory state of activation. Methods We measured the release of a panel of mediators produced in vitro by murine neutrophils and macrophages exposed to various biofilm components of P. aeruginosa cultures. Results We found that conditioned media from a high biofilm-producing strain of P. aeruginosa, PAR5, accumulated high concentrations of extracellular bacterial LPS, DNA and EPS by 72 h. These conditioned media induced phagocytes to release a hyperinflammatory pattern of mediators, with enhanced levels of $TNF-\alpha$, IL-6, IL12p40, $PGE_{2}$ and NO. Moreover, the phagocytes also upregulated COX-2 and iNOS with no influence on the expression of arginase-1. Conclusions Phagocytes exposed to biofilm microenvironment, called by us biofilm-associated neutrophils/macrophages (BANs/BAMs), display secretory properties similar to that of N1/M1-type phagocytes. These results suggest that in vivo high concentrations of LPS and DNA, trapped in biofilm by EPS, might convert infiltrating phagocytes into cells responsible for tissue injury without direct contact with bacteria and phagocytosis.

Mycobacterium tuberculosis (Mtb) is a highly efficient facultative intracellular pathogen circumventing attempts by the immune system to eradicate it. Alveolar macrophages are the primary target for intracellular invasion, and central to its pathogenesis is Mtb’s remarkable capacity to survive and proliferate within the hostile environment of these professional phagocytic cells. Despite its status as an intracellular pathogen, numerous extracellular bacteria can be found in caseating granulomas, the histopathological hallmark of TB disease. Mycobacterium marinum, the closest genetic relative to Mtb, thrives extracellularly in the absence of macrophages in zebrafish. Extracellular Mtb seen in histopathological lesions are likely to arise, at least in part, from macrophage necrosis which is associated with outer membrane damage and release of nuclear and cytoplasmic contents into the extracellular space. But several organisms, including other mycobacteria, have versatile strategies for escape into the extracellular environment, both by triggering cell lysis and leaving the host cell intact. The exit of intracellular organisms is a crucial stage of pathogenesis since cellular escape facilitates spread of infection to neighbouring cell, tissue invasion and onward transmission to new hosts. However, our understanding of the exit of Mtb from human macrophages is limited. Because studies examining mycobacterial control in various in vitro and in vivo models have not sought to quantify extracellular Mtb, the relative ratio and clinical relevance of this population to overall bacterial burden remains a matter of debate. In this study we use a novel high throughput flow cytometric assay based on quantitation of fluorescent Mtb to show that bacteria do not solely remain intracellular upon infection of macrophages, but a significant amount are located extracellularly. Our mathematical model to describe intracellular and extracellular bacillary accumulation predicts that bacteria must be accumulating in the extracellular space by means of a cytolysis independent strategy. We demonstrate the never previously described exit of Mtb from human macrophages by non-lytic egress and experimentally determine the rate at which this occurs. Non-lytic egress may be associated with virulence since we find that Mycobacterium Bovis bacillus Calmette-Guerin (BCG) does not exhibit the same behaviour.

We hypothesised that foetal immune responses to an infectious challenge may be detected by genome-wide transcriptional profiling of cord blood. In order to test this hypothesis, we sought to identify transcriptomic changes in post-natal cord blood samples following prolonged pre-labour rupture of membranes (PROM) as a surrogate for increased risk of infection. By comparison to controls we found increased levels of blood transcripts in a subset of prolonged PROM cases, significantly enriched for innate immune system signalling pathways. These changes were idiosyncratic, suggesting qualitative and quantitative variation in foetal immune responses which may reflect differences in exposure and/or in host genetics. Our data support the view that PROM represents an infection risk to the foetus. In addition, we propose that cord blood transcriptional profiling offers exciting opportunities to identify immune correlates of clinical outcome following potential in utero exposures to infection. These may be used to elucidate the mechanisms of immunological protection and pathology in the foetus and identify biomarkers to stratify the risk of adverse outcomes.

Urine-derived lymphocytes (UDLs) may serve as a non-invasive dynamic biomarker, reflective of the immune checkpoint phenotype and T-cell receptor repertoire of tumor-infiltrating lymphocytes. Patients with UDLs expressing high levels of PD-1 had worse clinical outcomes in muscle invasive bladder cancer., Despite the advances in cancer immunotherapy, only a fraction of patients with bladder cancer exhibit responses to checkpoint blockade, highlighting a need to better understand drug resistance and identify rational immunotherapy combinations. However, accessibility to the tumor prior and during therapy is a major limitation in understanding the immune tumor microenvironment (TME). Herein, we identified urine-derived lymphocytes (UDLs) as a readily accessible source of T cells in 32 patients with muscle invasive bladder cancer (MIBC). We observed that effector CD8+ and CD4+ cells and regulatory T cells within the urine accurately map the immune checkpoint landscape and T cell receptor repertoire of the TME. Finally, an increased UDL count, specifically high expression of PD-1 (PD-1hi) on CD8+ at the time of cystectomy, was associated with a shorter recurrence-free survival. UDL analysis represents a dynamic liquid biopsy that is representative of the bladder immune TME that may be used to identify actionable immuno-oncology (IO) targets with potential prognostic value in MIBC., Graphical Abstract

Clonal expansion of lymphocytes is a hallmark of vertebrate adaptive immunity. A small number of precursor cells that recognize a specific antigen proliferate into expanded clones, differentiate and acquire various effector and memory phenotypes, which promote effective immune responses. Recent studies establish a large degree of heterogeneity in the level of expansion and in cell state between and within expanding clones. Studying these processes in vivo, while providing insightful information on the level of heterogeneity, is challenging due to the complex microenvironment and the inability to continuously track individual cells over extended periods of time. Live cell imaging of ex vivo cultures within micro fabricated arrays provides an attractive methodology for studying clonal expansion. These experiments facilitate continuous acquisition of a large number of parameters on cell number, proliferation, death and differentiation state, with single-cell resolution on thousands of expanding clones that grow within controlled environments. Such data can reveal stochastic and instructive mechanisms that contribute to observed heterogeneity and elucidate the sequential order of differentiation events. Intercellular interactions can also be studied within these arrays by following responses of a controlled number of interacting cells, all trapped within the same microwell. Here we describe implementations of live-cell imaging within microwell arrays for studies of lymphocyte clonal expansion, portray insights already gained from these experiments and outline directions for future research. These tools, together with in vivo experiments tracking single-cell responses, will expand our understanding of adaptive immunity and the ways by which it can be manipulated.

Over the last century, scientists have embraced the idea of mobilizing antitumor immune responses in patients with cancer. In the last decade, we have seen the rebirth of cancer immunotherapy and its validation in a series of high profile clinical trials following the discovery of several immune-regulatory receptors. Recent studies point toward the tumor mutational load and resulting neoantigen burden as being crucial to tumor cell recognition by the immune system, highlighting a potentially targetable Achilles' heel in cancer. In this review, we explore the key mechanisms that underpin the recognition of cancerous cells by the immune system and discuss how we may advance immunotherapeutic strategies to target the cancer mutanome to stimulate tumor-specific immune responses, ultimately, to improve the clinical outcome for patients with cancer.

The T-cell receptor (TCR) repertoire is formed by random recombinations of genomic precursor elements; the resulting combinatorial diversity renders unlikely extensive TCR sharing between individuals. Here, we studied CDR3β amino acid sequence sharing in a repertoire-wide manner, using high-throughput TCR-seq in 28 healthy mice. We uncovered hundreds of public sequences shared by most mice. Public CDR3 sequences, relative to private sequences, are two orders of magnitude more abundant on average, express restricted V/J segments, and feature high convergent nucleic acid recombination. Functionally, public sequences are enriched for MHC-diverse CDR3 sequences that were previously associated with autoimmune, allograft, and tumor-related reactions, but not with anti-pathogen-related reactions. Public CDR3 sequences are shared between mice of different MHC haplotypes, but are associated with different, MHC-dependent, V genes. Thus, despite their random generation process, TCR repertoires express a degree of uniformity in their post-genomic organization. These results, together with numerical simulations of TCR genomic rearrangements, suggest that biases and convergence in TCR recombination combine with ongoing selection to generate a restricted subset of self-associated, public CDR3 TCR sequences, and invite reexamination of the basic mechanisms of T-cell repertoire formation.

The active vitamin D metabolite 1α,25-dihydroxyvitamin D (1,25[OH]2D) potently inhibits DC priming of T-cell activation, suggesting that it mediates a homeostatic role in this context. Therefore, careful regulation of 1,25[OH]2D levels is necessary to avoid inappropriate inhibition of T-cell activation. Cell-autonomous control of vitamin D activity can be modulated by the action of the vitamin D-activating and -inactivating hydroxylases, CYP27B1, and CYP24A1, respectively. We show that in comparison to macrophages, human monocyte-derived DCs exhibit significantly less activation of 25-dihydroxyvitamin D to 1,25[OH]2D, and that DCs predominantly express a truncated CYP27B1 transcript that may contribute to the deficiency in activation of vitamin D. Furthermore, in response to stimulation with 1,25[OH]2D, upregulation of the inactivating enzyme CYP24A1 curtailed the functional effects of vitamin D in DCs, but not macrophages. Production of 1,25[OH]2D by macrophages was adequate to induce expression of vitamin D-responsive genes by DCs, inhibit DC maturation in response to innate immune stimulation and DC-dependent T-cell responses. Our data suggest that in comparison to macrophages, differential regulation of hydroxylases limits autocrine vitamin D activity in DCs, and that paracrine activation of vitamin D exerts a more potent mechanism for homeostatic control of DC function.

Background ADATPeR is the first prospective study evaluating the role of anti-PD1 agents in the neoadjuvant setting prior to cytoreductive nephrectomy in treatment-naive patients with metastatic clear cell renal cell carcinoma (mccRCC). We performed multi-omic analyses to resolve spatial heterogeneity and temporal dynamics in putative biomarkers of response to anti-PD1 blockade. Methods In a single center study, patients received nivolumab (3mg/kg every 2 weeks) pre- and post-operatively until progressive disease (PD). Primary endpoint was safety, secondary endpoints were response evaluation and exploratory biomarker analysis. Multiregion tumour biopsies were obtained at baseline, on-treatment (week 9) and at PD. Whole-exome sequencing was performed to infer somatic mutations and predict candidate neoantigens (NAs). Tumour immune microenvironment was evaluated using a RNA-seq-derived immune signature and by stromal and intraepithelial tumour infiltrating lymphocytes (TILs) assessments. Results 15 patients were treated. At median follow-up of 12.5 months(m), nivolumab had an acceptable side-effect profile. Overall response rate was 37%. Preliminary transcriptome analyses of pre-treatment biopsies (33 samples from 14 patients; up to 4 regions per case) revealed enrichment for primary-resistance (defined as PD within 2m; n = 4) with immune ‘cold’ tumours, distinct from ’hot’ tumours. Histologic TILs scoring showed concordant immune phenotypic clusters. Primary-resistant cases demonstrated 0% on-treatment stromal- and IE-TILs (2 evaluable patients). In contrast, we observed heavy on-treatment stromal TILs (70-90%) and intraepithelial TILs (30-90%) across 7 regions at nephrectomy in an exceptional responder receiving ongoing treatment (>24 cycles). Conclusions ADATPeR is the first neoadjuvant immune checkpoint inhibitor study pre-cytoreductive nephrectomy, and incorporated multi-omic analyses of putative biomarkers. Baseline immune gene expression signature is distinct in responders compared with non-responders. On-treatment intraepithelial TILs were prominent in those deriving durable clinical benefit. Integrative analyses are ongoing. Clinical trial identification NCT02446860. Legal entity responsible for the study The authors. Funding Bristol-Myers Squibb; The National Institute for Health Research (NIHR) Biomedical Research Centre (BRC) at The Royal Marsden NHS Foundation Trust and The Institute of Cancer Research; Cancer Research UK (CRUK). Disclosure All authors have declared no conflicts of interest.

2590 Background: CD4 T helper cells are key orchestrators of immunity in states of persistent antigen exposure. In chronic viral infection, loss of immune control is associated with CD4 differentiation skewing (CD4ds) resulting from decline of early progenitors and gain in abundance of exhausted and terminally differentiated subsets. Here, we set out to identify whether a similar process occurs within the tumour microenvironment, contributing to immune dysfunction. Methods: Multiregional samples of tumour and non-tumour lung tissue from patients with untreated, surgically resected non-small cell lung cancer (NSCLC) within the first 100 recruited to the prospective lung TRACERx study were analysed by high dimensional flow cytometry of tumour infiltrating lymphocytes (TILs) and paired bulk tumour exome and RNA sequencing. We additionally reanalysed publically available single T cell and bulk tumour RNA sequencing from patients with NSCLC. Results: Unsupervised clustering and dimension reduction revealed a heterogenous landscape of CD4 TILs, with evidence of CD4ds in association with tumour mutational burden. Loss of PD1-CCR7+ T central memory enriched early differentiated cells was accompanied by gain in abundance of PD1+ populations with exhausted (CD57-ICOShiCTLA4hi) and terminally differentiated effector (CD57+Eomes+) phenotypes. Further characterisation of these populations by single cell RNA sequencing revealed differential expression of key genes involved in transcriptional regulation, co-inhibition and co-stimulatory pathways. A validated gene signature of CD4ds was associated with worse outcomes in TRACERx and TCGA cohorts. Conclusions: Our findings reveal remodelling of the CD4 differentiation landscape in association with tumour genomic characteristics, underscoring how mutational burden in the context of chronic stimulation may lead to loss of immune fitness and elucidating key regulatory pathways in potentially clinically relevant CD4 subsets.

T cells integrate cell-specific Ag receptor signaling with shared signals mediated by secreted cytokines, which often involve regulatory feedback loops. IL-2 signaling, for example, reduces the synthesis of IL-2 and increases the synthesis of IL-2Rα-chain, whereas both genes require TCR signaling for their activation. The ways by which T cells dynamically integrate these private and public signals during activation are not well understood. We combined robotics, multiparameter flow cytometry, and real-time quantitative PCR to analyze T cell activation at high temporal resolution over several days. Two distinct temporal phases of T cell activation were evident. First, Ag-dependent signals activated low IL-2Rα and high IL-2 production, independent of IL-2 signaling. Subsequently, secreted IL-2 acted as a shared resource driving high IL-2Rα expression, reduced IL-2 synthesis, and cell proliferation. This transition was independent of continued TCR signaling. Our data allowed the determination of the parameters of the IL-2–mediated extracellular positive and negative feedback circuits and demonstrated that the two loops are coupled and become activated at a similar level of IL-2 signaling. We propose that temporal separation of private and shared signals allows T cells to first integrate Ag-specific responses and subsequently share information leading to collective decision making.

Summary: High-throughput sequencing provides an opportunity to analyse the repertoire of antigen-specific receptors with an unprecedented breadth and depth. However, the quantity of raw data produced by this technology requires efficient ways to categorize and store the output for subsequent analysis. To this end, we have defined a simple five-item identifier that uniquely and unambiguously defines each TcR sequence. We then describe a novel application of finite-state automaton to map Illumina short-read sequence data for individual TcRs to their respective identifier. An extension of the standard algorithm is also described, which allows for the presence of single-base pair mismatches arising from sequencing error. The software package, named Decombinator, is tested first on a set of artificial in silico sequences and then on a set of published human TcR-β sequences. Decombinator assigned sequences at a rate more than two orders of magnitude faster than that achieved by classical pairwise alignment algorithms, and with a high degree of accuracy (>88%), even after introducing up to 1% error rates in the in silico sequences. Analysis of the published sequence dataset highlighted the strong V and J usage bias observed in the human peripheral blood repertoire, which seems to be unconnected to antigen exposure. The analysis also highlighted the enormous size of the available repertoire and the challenge of obtaining a comprehensive description for it. The Decombinator package will be a valuable tool for further in-depth analysis of the T-cell repertoire. Availability and implementation: The Decombinator package is implemented in Python (v2.6) and is freely available at https://github.com/uclinfectionimmunity/Decombinator along with full documentation and examples of typical usage. Contact: b.chain@ucl.ac.uk

Modeling memory differentiation in T cells The balance between effector and central memory T cells shifts toward the latter as the number of T cells participating in immune responses increases. Polonsky et al. determined the mechanisms by which T cell quorum sensing affects memory differentiation by using live-cell imaging to track cell proliferation and differentiation. They found that the rate of memory CD4 + T cell differentiation is determined by cell number. This rate substantially increases above a threshold number of locally interacting cells. Mathematical modeling suggests that the number of initially seeded cells and the number of cell divisions are not critical. Instead, the instantaneous number of interacting cells continuously modulates the differentiation rate. This is partly fueled by increased sensitivity to the cytokines interleukin-2 (IL-2) and IL-6, independent of any effects on cell proliferation. Science , this issue p. eaaj1853

Numerous gene signatures, or modules have been described to evaluate the immune cell composition in transcriptomes of multicellular tissue samples. However, significant diversity in module gene content for specific cell types is associated with heterogeneity in their performance. In order to rank modules that best reflect their purported association, we have generated the modular discrimination index (MDI) score that assesses expression of each module in the target cell type relative to other cells. We demonstrate that MDI scores predict modules that best reflect independently validated differences in cellular composition, and correlate with the covariance between cell numbers and module expression in human blood and tissue samples. Our analyses demonstrate that MDI scores provide an ordinal summary statistic that reliably ranks the accuracy of gene expression modules for deconvolution of cell type abundance in transcriptional data.

Eukaryotic cells have the ability to uptake and transport endogenous and exogenous DNA in their nuclei, however little is known about the specific pathways involved. Here we show that the nuclear transport receptor importin 7 (imp7) supports nuclear import of supercoiled plasmid DNA and human mitochondrial DNA in a Ran and energy-dependent way. The imp7-dependent pathway was specifically competed by excess DNA but not by excess of maltose-binding protein fused with the classical nuclear localizing signal (NLS) or the M9 peptides. Transport of DNA molecules complexed with poly-l-lysine was impaired in intact cells depleted of imp7, and DNA complexes remained localized in the cytoplasm. Poor DNA nuclear import in cells depleted of imp7 directly correlated with lower gene expression levels in these cells compared to controls. Inefficient nuclear import of transfected DNA induced greater upregulation of the interferon pathway, suggesting that rapid DNA nuclear import may prevent uncontrolled activation of the innate immune response. Our results provide evidence that imp7 is a non-redundant component of an intrinsic pathway in mammalian cells for efficient accumulation of exogenous and endogenous DNA in the nucleus, which may be critical for the exchange of genetic information between mitochondria and nuclear genomes and to control activation of the innate immune response.

Dendritic cells (DCs) are an attractive target for DNA vaccines as they are potent antigen presenting cells. This study demonstrated how non-viral gene delivery to DCs involving complexes of poly-l-lysine (PLL) and plasmid DNA (pDNA) (polyplexes) showed dependence on DNA vector topology. DNA topology is of importance from both production and regulatory viewpoints. In our previous study with CHO cells we demonstrated that polyplex uptake was dependent on DNA topology whereby complexes containing supercoiled (SC) pDNA were smaller, more resistant to nucleases and more effectively condensed by PLL than open circular (OC) and linear-pDNA complexes.In this study polyplex uptake in DCs was measured qualitatively and quantitatively by confocal microscopy along with gene expression studies and measurement of DC phenotype. PLL is known for its ability to condense DNA and serve as an effective gene delivery vehicle. Quantification studies revealed that by 1h following uptake 15% (±2.59% relative standard error [RSE]) of SC-pDNA polyplexes were identified to be associated (fluorescent co-localisation) with the nucleus, in comparison to no nuclear association identified for OC- and linear-pDNA complexes. By 48h following uptake, 30% (±1.82% RSE) of SC-pDNA complexes associated with the nucleus in comparison to 16% (±4.40% RSE) and 12% (±6.97% RSE) of OC- and linear-pDNA polyplexes respectively. Confocal microscopy images showed how DNA and PLL remained associated following uptake by dual labelling. Polyplex (containing 20μg pDNA) gene expression (plasmid encoded lacZ [β-galactosidase] reporter gene) in DCs was greatest for SC-pDNA polyplexes at 14.12% unlike that of OC- (9.59%) and linear-pDNA (7.43%). DCs express cell surface markers which contribute towards antigen presentation. Polyplex gene expression did not alter DC phenotype through surface marker expression. This may be due to the pDNA dose employed (20μg) as other studies have used doses as high as 200μg pDNA to induce DC phenotypic changes. Although no change in DC phenotype occurred, this could be advantageous in terms of biocompatibility. Collectively these results indicate that DNA topology is an important parameter for DC vector design, particularly pDNA in the SC conformation in regards to DNA vaccination studies.