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Concerted efforts aim to reduce the burden of 6 months of anti-tuberculous treatment for tuberculosis (TB). Treatment cessation at 8 weeks is effective for most but incurs increased risk of disease relapse. We tested the hypothesis that blood RNA signatures or C-reactive protein (CRP) measurements discriminate 8-week sputum culture status, as a prerequisite for a biomarker to stratify risk of relapse following treatment cessation at this time-point.
BackgroundThe relationship between tuberculosis (TB), one of the leading infectious causes of death worldwide, and the microbiome, which is critical for health, is poorly understood.MethodsTo identify potential microbiome-host interactions, profiling of the oral, sputum and stool microbiota [n = 58 cases, n = 47 culture-negative symptomatic controls (SCs)] and whole blood transcriptome were done in pre-treatment presumptive pulmonary TB patients. This was a cross-sectional study. Microbiota were also characterised in close contacts of cases (CCCs, n = 73) and close contacts of SCs (CCSCs, n = 82) without active TB.FindingsCases and SCs each had similar α- and β-diversities in oral washes and sputum, however, β-diversity differed in stool (PERMANOVA p = 0•035). Cases were enriched with anaerobes in oral washes, sputum (Paludibacter, Lautropia in both) and stool (Erysipelotrichaceae, Blautia, Anaerostipes) and their stools enriched in microbial genes annotated as amino acid and carbohydrate metabolic pathways. In pairwise comparisons with their CCCs, cases had Megasphaera-enriched oral and sputum microbiota and Bifidobacterium-, Roseburia-, and Dorea-depleted stools. Compared to their CCSCs, SCs had reduced α-diversities and many differential taxa per specimen type. Cases differed transcriptionally from SCs in peripheral blood (PERMANOVA p = 0•001). A co-occurrence network analysis showed stool taxa, Erysipelotrichaceae and Blautia, to negatively co-correlate with enriched "death receptor" and "EIF2 signalling" pathways whereas Anaerostipes positively correlated with enriched "interferon signalling", "Nur77 signalling" and "inflammasome" pathways; all of which are host pathways associated with disease severity. In contrast, none of the taxa enriched in SCs correlated with host pathways.InterpretationTB-specific microbial relationships were identified in oral washes, induced sputum, and stool from cases before the confounding effects of antibiotics. Specific anaerobes in cases' stool predict upregulation of pro-inflammatory immunological pathways, supporting the gut microbiota's role in TB.FundingEuropean & Developing Countries Clinical Trials Partnership, South African-Medical Research Council, National Institute of Allergy and Infectious Diseases.
Objective In people living with HIV (PLHIV), we sought to test the hypothesis that long term anti-retroviral therapy restores the normal T cell repertoire, and investigate the functional relationship of residual repertoire abnormalities to persistent immune system dysregulation. Methods We conducted a case-control study in PLHIV and HIV-negative volunteers, of circulating T cell receptor repertoires and whole blood transcriptomes by RNA sequencing, complemented by metadata from routinely collected health care records. Results T cell receptor sequencing revealed persistent abnormalities in the clonal T cell repertoire of PLHIV, characterized by reduced repertoire diversity and oligoclonal T cell expansion correlated with elevated CD8 T cell counts. We found no evidence that these expansions were driven by cytomegalovirus or another common antigen. Increased frequency of long CDR3 sequences and reduced frequency of public sequences among the expanded clones implicated abnormal thymic selection as a contributing factor. These abnormalities in the repertoire correlated with systems level evidence of persistent T cell activation in genome-wide blood transcriptomes. Conclusions The diversity of T cell receptor repertoires in PLHIV on long term anti-retroviral therapy remains significantly depleted, and skewed by idiosyncratic clones, partly attributable to altered thymic output and associated with T cell mediated chronic immune activation. Further investigation of thymic function and the antigenic drivers of T cell clonal selection in PLHIV are critical to efforts to fully re-establish normal immune function.
The T cell receptor (TCR) repertoire can provide a personalised biomarker for infectious and non-infectious diseases. We describe a protocol for amplifying, sequencing and analysing TCRs which is robust, sensitive and versatile. The key experimental step is ligation of a single stranded oligonucleotide to the 3' end of the TCR cDNA. This allows amplification of all possible rearrangements using a single set of primers per locus. It also introduces a unique molecular identifier to label each starting cDNA molecule. This molecular identifier is used to correct for sequence errors and for effects of differential PCR amplification efficiency, thus producing more accurate measures of the true TCR frequency within the sample. This integrated experimental and computational pipeline is applied to the analysis of human memory and naive subpopulations, and results in consistent measures of diversity and inequality. After error correction, the distribution of TCR sequence abundance in all subpopulations followed a power law over a wide range of values. The power law exponent differed between naïve and memory populations, but was consistent between individuals. The integrated experimental and analysis pipeline we describe is appropriate to studies of T cell responses in a broad range of physiological and pathological contexts.
Approximately 10% of patients hospitalised with COVID-19 have residual lung abnormalities (RLAs) at 12 months. In clinical practice, CT scan appearances are often used to guide management. Whether such radiological changes reliably reflect immunopathomechanisms, and can therefore inform the treatment approach, is unclear and an important clinical question.
Methods
We compared the single cell transcriptomic and T cell receptor (TCR) profiles of bronchoalveolar lavage cells from patients with Post-COVID RLAs with either predominantly inflammatory or fibrotic radiological appearances.
Results
We generated a dataset of 55, 776 cells. CD4 central memory T cells (TCM) and CD8 effector memory T cells (TEM) were significantly increased in the inflammatory sub-phenotype. Both patient groups were transcriptionally similar and exhibited clonal expansion and high TCR clustering, without enrichment for SARS-CoV-2 reactive sequences.
Conclusions
We describe the first comparison of purported radiological subphenotypes in patients with Post-COVID RLAs, which may actually represent different manifestations of the same disease. Antigen-specific immune responses to unidentified T cell targets, imply a breach of immune tolerance in the lung and a potential role for T-cell directed therapies in these patients, agonistic of radiological appearance. Please refer to page A285 for declarations of interest related to this abstract.
Background Persistent radiological lung abnormalities are evident in many survivors of acute coronavirus disease 2019 (COVID-19). Consolidation and ground glass opacities are interpreted to indicate subacute inflammation whereas reticulation is thought to reflect fibrosis. We sought to identify differences at molecular and cellular level, in the local immunopathology of post-COVID inflammation and fibrosis. Methods We compared single-cell transcriptomic profiles and T cell receptor (TCR) repertoires of bronchoalveolar cells obtained from convalescent individuals with each radiological pattern, targeting lung segments affected by the predominant abnormality. Results CD4 central memory T cells and CD8 effector memory T cells were significantly more abundant in those with inflammatory radiology. Clustering of similar TCRs from multiple donors was a striking feature of both phenotypes, consistent with tissue localised antigen-specific immune responses. There was no enrichment for known SARS-CoV-2-reactive TCRs, raising the possibility of T cell-mediated immunopathology driven by failure in immune self-tolerance. Conclusions Post-COVID radiological inflammation and fibrosis show evidence of shared antigen-specific T cell responses, suggesting a role for therapies targeting T cells in limiting post-COVID lung damage.
Abstract 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. Interferon gamma (IFNγ) responses do not discriminate between protection and pathogenicity, but IL-17A/F responses, known to drive pathology in diverse chronic inflammatory diseases, have also been associated with TB pathogenesis in animal models. At the site of in vivo immune recall responses to Mtb modelled by the tuberculin skin test, we show for the first time that active TB in humans is also associated with exaggerated IL-17A/F expression, accumulation of Th17 cells and IL-17A/F 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. Our data support targeting of this pathway in host-directed therapy for TB.
Rationale: There is poor understanding about protective immunity and the pathogenesis of cavitation in patients with tuberculosis.Objectives: To map pathophysiological pathways at anatomically distinct positions within the human tuberculosis cavity.Methods: Biopsies were obtained from eight predetermined locations within lung cavities of patients with multidrug-resistant tuberculosis undergoing therapeutic surgical resection (n = 14) and healthy lung tissue from control subjects without tuberculosis (n = 10). RNA sequencing, immunohistochemistry, and bacterial load determination were performed at each cavity position. Differentially expressed genes were normalized to control subjects without tuberculosis, and ontologically mapped to identify a spatially compartmentalized pathophysiological map of the cavity. In silico perturbation using a novel distance-dependent dynamical sink model was used to investigate interactions between immune networks and bacterial burden, and to integrate these identified pathways.Measurements and Main Results: The median (range) lung cavity volume on positron emission tomography/computed tomography scans was 50 cm3 (15–389 cm3). RNA sequence reads (31% splice variants) mapped to 19,049 annotated human genes. Multiple proinflammatory pathways were upregulated in the cavity wall, whereas a downregulation "sink" in the central caseum–fluid interface characterized 53% of pathways including neuroendocrine signaling, calcium signaling, triggering receptor expressed on myeloid cells-1, reactive oxygen and nitrogen species production, retinoic acid–mediated apoptosis, and RIG-I-like receptor signaling. The mathematical model demonstrated that neuroendocrine, protein kinase C-θ, and triggering receptor expressed on myeloid cells-1 pathways, and macrophage and neutrophil numbers, had the highest correlation with bacterial burden (r > 0.6), whereas T-helper effector systems did not.Conclusions: These data provide novel insights into host immunity to Mycobacterium tuberculosis–related cavitation. The pathways defined may serve as useful targets for the design of host-directed therapies, and transmission prevention interventions.
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