Blood and Site of Disease Inflammatory Profiles Differ in Patients With Pericardial Tuberculosis and Human Immunodeficiency Virus Type 1.

Background: To better understand the pathogenesis of pericardial tuberculosis (PCTB), we sought to characterize the systemic inflammatory profile in people with human immunodeficiency virus type 1 (HIV-1) with latent TB infection (LTBI), pulmonary TB (PTB), or PCTB. Methods: Using Luminex, we measured the concentration of 39 analytes in pericardial fluid (PCF) and paired plasma from 18 PCTB participants, and plasma from 16 LTBI and 20 PTB participants. Follow-up plasma samples were also obtained from PTB and PCTB participants. HLA-DR expression on Mycobacterium tuberculosis-specific CD4 T cells was measured in baseline samples using flow cytometry. Results: Assessment of the overall systemic inflammatory profile by principal component analysis showed that the inflammatory profile of active TB participants was distinct from the LTBI group, while PTB patients could not be distinguished from those with PCTB. When comparing the inflammatory profile between PCF and paired blood, we found that the concentrations of most analytes (25/39) were elevated at site of disease. However, the inflammatory profile in PCF partially mirrored inflammatory events in the blood. After TB treatment completion, the overall plasma inflammatory profile reverted to that observed in the LTBI group. Lastly, HLA-DR expression showed the best performance for TB diagnosis compared to previously described biosignatures built from soluble markers. Conclusions: Our results show that the inflammatory profile in blood was comparable between PTB and PCTB. However, at the site of infection (PCF), inflammation was significantly elevated compared to blood. Additionally, our data emphasize the potential role of HLA-DR expression as a biomarker for TB diagnosis.

Corrigendum: Comparison of the frequency and phenotypic profile of Mycobacterium tuberculosis-specific CD4 T cells between the site of disease and blood in pericardial tuberculosis.

[This corrects the article DOI: 10.3389/fimmu.2022.1009016.].

Phenotypic Profile of Mycobacterium tuberculosis-Specific CD4 T-Cell Responses in People With Advanced Human Immunodeficiency Virus Who Develop Tuberculosis-Associated Immune Reconstitution Inflammatory Syndrome.

Background: Tuberculosis-associated immune reconstitution inflammatory syndrome (TB-IRIS) is a frequent complication of cotreatment for TB and human immunodeficiency virus (HIV)-1. We characterized Mycobacterium tuberculosis (Mtb)-specific CD4 T-cell phenotype and transcription factor profile associated with the development of TB-IRIS. Methods: We examined the role of CD4 T-cell transcription factors in a murine model of mycobacterial IRIS. In humans, we used a longitudinal study design to compare the magnitude of antiretroviral therapy, activation, transcription factor profile, and cytotoxic potential of Mtb-specific CD4 T cells between TB-IRIS (n = 25) and appropriate non-IRIS control patients (n = 18) using flow cytometry. Results: In the murine model, CD4 T-cell expression of Eomesodermin (Eomes), but not Tbet, was associated with experimentally induced IRIS. In patients, TB-IRIS onset was associated with the expansion of Mtb-specific IFNγ+CD4 T cells (P = .039). Patients with TB-IRIS had higher HLA-DR expression (P = .016), but no differences in the expression of T-bet or Eomes were observed. At TB-IRIS onset, Eomes+Tbet+Mtb-specific IFNγ+CD4+ T cells showed higher expression of granzyme B in patients with TB-IRIS (P = .026). Conclusions: Although the murine model of Mycobacterium avium complex-IRIS suggests that Eomes+CD4 T cells underly IRIS, TB-IRIS was not associated with Eomes expression in patients. Mycobacterium tuberculosis-specific IFNγ+CD4 T-cell responses in TB-IRIS patients are differentiated, highly activated, and potentially cytotoxic.

Effects of tuberculosis and/or HIV-1 infection on COVID-19 presentation and immune response in Africa.

Few studies from Africa have described the clinical impact of co-infections on SARS-CoV-2 infection. Here, we investigate the presentation and outcome of SARS-CoV-2 infection in an African setting of high HIV-1 and tuberculosis prevalence by an observational case cohort of SARS-CoV-2 patients. A comparator group of non SARS-CoV-2 participants is included. The study includes 104 adults with SARS-CoV-2 infection of whom 29.8% are HIV-1 co-infected. Two or more co-morbidities are present in 57.7% of participants, including HIV-1 (30%) and active tuberculosis (14%). Amongst patients dually infected by tuberculosis and SARS-CoV-2, clinical features can be typical of either SARS-CoV-2 or tuberculosis: lymphopenia is exacerbated, and some markers of inflammation (D-dimer and ferritin) are further elevated (p < 0.05). Amongst HIV-1 co-infected participants those with low CD4 percentage strata exhibit reduced total, but not neutralising, anti-SARS-CoV-2 antibodies. SARS-CoV-2 specific CD8 T cell responses are present in 35.8% participants overall but undetectable in combined HIV-1 and tuberculosis. Death occurred in 30/104 (29%) of all COVID-19 patients and in 6/15 (40%) of patients with coincident SARS-CoV-2 and tuberculosis. This shows that in a high incidence setting, tuberculosis is a common co-morbidity in patients admitted to hospital with COVID-19. The immune response to SARS-CoV-2 is adversely affected by co-existent HIV-1 and tuberculosis.

Comparison of the frequency and phenotypic profile of Mycobacterium tuberculosis-specific CD4 T cells between the site of disease and blood in pericardial tuberculosis.

Studies of the immune response at the site of disease in extra-pulmonary tuberculosis (EPTB) disease are scarce. In this study, we compared the cellular profile of Mycobacterium tuberculosis (Mtb)-specific T cells in pericardial fluid and peripheral blood in patients with pericardial TB (PCTB). Whole blood and pericardial fluid (PCF) samples were collected at the time of diagnostic sampling, with repeat blood sampling after completion of anti-tubercular treatment (ATT) in 16 PCTB patients, most of them being HIV-1 infected (n=14). These samples were stimulated ex vivo and the phenotypic and functional cellular profile of PCF and blood was assessed by flow cytometry. We found that lymphocytes were the predominant cell type in PCF in PCTB, with a preferential influx of CD4 T cells. The frequencies of TNF-α producing Mtb-specific granulocytes and Mtb-specific CD4 T cells were significantly higher in PCF compared to blood. Mtb-specific CD4 T cells in PCF exhibited a distinct phenotype compared to those in blood, with greater GrB expression and lower CD27 and KLRG1 expression. We observed no difference in the production IFNγ, TNF or IL-2 by Mtb-specific CD4 T cells between the two compartments, but MIP-1ß production was lower in the PCF T cells. Bacterial loads were not associated with alterations in the phenotype or function of Mtb-specific CD4 T cells. Upon ATT completion, HLA-DR, Ki-67 and GrB expression was significantly decreased, and relative IL-2 production was increased in peripheral Mtb-specific CD4 T cells. Overall, using an ex vivo assay to compare the immune response towards Mtb in PCF and in blood, we identified significant difference in the phenotypic profile of Mtb-specific CD4 T response between these two compartments. Moreover, we show that the activation profile of peripheral Mtb-specific CD4 T cells could be used to monitor treatment response in PCTB.

GPX4 regulates cellular necrosis and host resistance in Mycobacterium tuberculosis infection.

Cellular necrosis during Mycobacterium tuberculosis (Mtb) infection promotes both immunopathology and bacterial dissemination. Glutathione peroxidase-4 (Gpx4) is an enzyme that plays a critical role in preventing iron-dependent lipid peroxidation-mediated cell death (ferroptosis), a process previously implicated in the necrotic pathology seen in Mtb-infected mice. Here, we document altered GPX4 expression, glutathione levels, and lipid peroxidation in patients with active tuberculosis and assess the role of this pathway in mice genetically deficient in or overexpressing Gpx4. We found that Gpx4-deficient mice infected with Mtb display substantially increased lung necrosis and bacterial burdens, while transgenic mice overexpressing the enzyme show decreased bacterial loads and necrosis. Moreover, Gpx4-deficient macrophages exhibited enhanced necrosis upon Mtb infection in vitro, an outcome suppressed by the lipid peroxidation inhibitor, ferrostatin-1. These findings provide support for the role of ferroptosis in Mtb-induced necrosis and implicate the Gpx4/GSH axis as a target for host-directed therapy of tuberculosis.

Histone acetylome-wide associations in immune cells from individuals with active Mycobacterium tuberculosis infection.

Host cell chromatin changes are thought to play an important role in the pathogenesis of infectious diseases. Here we describe a histone acetylome-wide association study (HAWAS) of an infectious disease, on the basis of genome-wide H3K27 acetylation profiling of peripheral blood granulocytes and monocytes from persons with active Mycobacterium tuberculosis (Mtb) infection and healthy controls. We detected >2,000 differentially acetylated loci in either cell type in a Singapore Chinese discovery cohort (n = 46), which were validated in a subsequent multi-ethnic Singapore cohort (n = 29), as well as a longitudinal cohort from South Africa (n = 26), thus demonstrating that HAWAS can be independently corroborated. Acetylation changes were correlated with differential gene expression. Differential acetylation was enriched near potassium channel genes, including KCNJ15, which modulates apoptosis and promotes Mtb clearance in vitro. We performed histone acetylation quantitative trait locus (haQTL) analysis on the dataset and identified 69 candidate causal variants for immune phenotypes among granulocyte haQTLs and 83 among monocyte haQTLs. Our study provides proof-of-principle for HAWAS to infer mechanisms of host response to pathogens.

T cell responses to SARS-CoV-2 spike cross-recognize Omicron.

The SARS-CoV-2 Omicron variant (B.1.1.529) has multiple spike protein mutations1,2 that contribute to viral escape from antibody neutralization3-6 and reduce vaccine protection from infection7,8. The extent to which other components of the adaptive response such as T cells may still target Omicron and contribute to protection from severe outcomes is unknown. Here we assessed the ability of T cells to react to Omicron spike protein in participants who were vaccinated with Ad26.CoV2.S or BNT162b2, or unvaccinated convalescent COVID-19 patients (n = 70). Between 70% and 80% of the CD4+ and CD8+ T cell response to spike was maintained across study groups. Moreover, the magnitude of Omicron cross-reactive T cells was similar for Beta (B.1.351) and Delta (B.1.617.2) variants, despite Omicron harbouring considerably more mutations. In patients who were hospitalized with Omicron infections (n = 19), there were comparable T cell responses to ancestral spike, nucleocapsid and membrane proteins to those in patients hospitalized in previous waves dominated by the ancestral, Beta or Delta variants (n = 49). Thus, despite extensive mutations and reduced susceptibility to neutralizing antibodies of Omicron, the majority of T cell responses induced by vaccination or infection cross-recognize the variant. It remains to be determined whether well-preserved T cell immunity to Omicron contributes to protection from severe COVID-19 and is linked to early clinical observations from South Africa and elsewhere9-12.

Baseline IL-6 is a biomarker for unfavourable tuberculosis treatment outcomes: a multisite discovery and validation study.

BACKGROUND: Biomarkers of unfavourable tuberculosis (TB) treatment outcomes are needed to accelerate new drug and regimen development. Whether plasma cytokine levels can predict unfavourable TB treatment outcomes is unclear. METHODS: We identified and internally validated the association between 20 a priori selected plasma inflammatory markers and unfavourable treatment outcomes of failure, recurrence and all-cause mortality among adults with drug-sensitive pulmonary TB in India. We externally validated these findings in two independent cohorts of predominantly diabetic and HIV co-infected TB patients in India and South Africa, respectively. RESULTS: Pre-treatment interferon-γ, interleukin (IL)-13 and IL-6 were associated with treatment failure in the discovery analysis. Internal validation confirmed higher pre-treatment IL-6 concentrations among failure cases compared with controls. External validation among predominantly diabetic TB patients found an association between pre-treatment IL-6 concentrations and subsequent recurrence and death. Similarly, external validation among predominantly HIV co-infected TB patients found an association between pre-treatment IL-6 concentrations and subsequent treatment failure and death. In a pooled analysis of 363 TB cases from the Indian and South African validation cohorts, high pre-treatment IL-6 concentrations were associated with higher risk of failure (adjusted OR (aOR) 2.16, 95% CI 1.08-4.33; p=0.02), recurrence (aOR 5.36, 95% CI 2.48-11.57; p<0.001) and death (aOR 4.62, 95% CI 1.95-10.95; p<0.001). Adding baseline IL-6 to a risk prediction model comprised of low body mass index, high smear grade and cavitation improved model performance by 15% (C-statistic 0.66 versus 0.76; p=0.02). CONCLUSIONS: Pre-treatment IL-6 is a biomarker for unfavourable TB treatment outcomes. Future studies should identify optimal IL-6 concentrations for point-of-care risk prediction.

Rapid, simplified whole blood-based multiparameter assay to quantify and phenotype SARS-CoV-2-specific T-cells.

BACKGROUND: Rapid tests to evaluate severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-specific T-cell responses are urgently needed to decipher protective immunity and aid monitoring vaccine-induced immunity. METHODS: Using a rapid whole blood assay requiring a minimal amount of blood, we measured qualitatively and quantitatively SARS-CoV-2-specific CD4 T-cell responses in 31 healthcare workers using flow cytometry. RESULTS: 100% of COVID-19 convalescent participants displayed a detectable SARS-CoV-2-specific CD4 T-cell response. SARS-CoV-2-responding cells were also detected in 40.9% of participants with no COVID-19-associated symptoms or who tested PCR-negative. Phenotypic assessment indicated that, in COVID-19 convalescent participants, SARS-CoV-2 CD4 responses displayed an early differentiated memory phenotype with limited capacity to produce interferon (IFN)-γ. Conversely, in participants with no reported symptoms, SARS-CoV-2 CD4 responses were enriched in late differentiated cells, coexpressing IFN-γ and tumour necrosis factor-α and also Granzyme B. CONCLUSIONS: This proof-of-concept study presents a scalable alternative to peripheral blood mononuclear cell-based assays to enumerate and phenotype SARS-CoV-2-responding T-cells, thus representing a practical tool to monitor adaptive immunity due to natural infection or vaccine trials.

Escape from recognition of SARS-CoV-2 variant spike epitopes but overall preservation of T cell immunity.

SARS-CoV-2 variants that escape neutralization and potentially affect vaccine efficacy have emerged. T cell responses play a role in protection from reinfection and severe disease, but the potential for spike mutations to affect T cell immunity is incompletely understood. We assessed neutralizing antibody and T cell responses in 44 South African COVID-19 patients either infected with the Beta variant (dominant from November 2020 to May 2021) or infected before its emergence (first wave, Wuhan strain) to provide an overall measure of immune evasion. We show that robust spike-specific CD4 and CD8 T cell responses were detectable in Beta-infected patients, similar to first-wave patients. Using peptides spanning the Beta-mutated regions, we identified CD4 T cell responses targeting the wild-type peptides in 12 of 22 first-wave patients, all of whom failed to recognize corresponding Beta-mutated peptides. However, responses to mutated regions formed only a small proportion (15.7%) of the overall CD4 response, and few patients (3 of 44) mounted CD8 responses that targeted the mutated regions. Among the spike epitopes tested, we identified three epitopes containing the D215, L18, or D80 residues that were specifically recognized by CD4 T cells, and their mutated versions were associated with a loss of response. This study shows that despite loss of recognition of immunogenic CD4 epitopes, CD4 and CD8 T cell responses to Beta are preserved overall. These observations may explain why several vaccines have retained the ability to protect against severe COVID-19 even with substantial loss of neutralizing antibody activity against Beta.

Eosinophils are part of the granulocyte response in tuberculosis and promote host resistance in mice.

Host resistance to Mycobacterium tuberculosis (Mtb) infection requires the activities of multiple leukocyte subsets, yet the roles of the different innate effector cells during tuberculosis are incompletely understood. Here we uncover an unexpected association between eosinophils and Mtb infection. In humans, eosinophils are decreased in the blood but enriched in resected human tuberculosis lung lesions and autopsy granulomas. An influx of eosinophils is also evident in infected zebrafish, mice, and nonhuman primate granulomas, where they are functionally activated and degranulate. Importantly, using complementary genetic models of eosinophil deficiency, we demonstrate that in mice, eosinophils are required for optimal pulmonary bacterial control and host survival after Mtb infection. Collectively, our findings uncover an unexpected recruitment of eosinophils to the infected lung tissue and a protective role for these cells in the control of Mtb infection in mice.

Relationship of SARS-CoV-2-specific CD4 response to COVID-19 severity and impact of HIV-1 and tuberculosis coinfection.

T cells are involved in control of coronavirus disease 2019 (COVID-19), but limited knowledge is available on the relationship between antigen-specific T cell response and disease severity. Here, we used flow cytometry to assess the magnitude, function, and phenotype of SARS coronavirus 2-specific (SARS-CoV-2-specific) CD4+ T cells in 95 hospitalized COVID-19 patients, 38 of them being HIV-1 and/or tuberculosis (TB) coinfected, and 38 non-COVID-19 patients. We showed that SARS-CoV-2-specific CD4+ T cell attributes, rather than magnitude, were associated with disease severity, with severe disease being characterized by poor polyfunctional potential, reduced proliferation capacity, and enhanced HLA-DR expression. Moreover, HIV-1 and TB coinfection skewed the SARS-CoV-2 T cell response. HIV-1-mediated CD4+ T cell depletion associated with suboptimal T cell and humoral immune responses to SARS-CoV-2, and a decrease in the polyfunctional capacity of SARS-CoV-2-specific CD4+ T cells was observed in COVID-19 patients with active TB. Our results also revealed that COVID-19 patients displayed reduced frequency of Mycobacterium tuberculosis-specific CD4+ T cells, with possible implications for TB disease progression. These results corroborate the important role of SARS-CoV-2-specific T cells in COVID-19 pathogenesis and support the concept of altered T cell functions in patients with severe disease.

TBDBT: A TB DataBase Template for collection of harmonized TB clinical research data in REDCap, facilitating data standardisation for inter-study comparison and meta-analyses.

Clinical tuberculosis research, both within research groups and across research ecosystems, is often undertaken in isolation using bespoke data collection platforms and applying differing data conventions. This failure to harmonise clinical phenotype data or apply standardised data collection and storage standards in turn limits the opportunity to undertake meta-analyses using data generated across multiple research projects for the same research domain. We have developed the Tuberculosis DataBase Template (TBDBT), a template for the well-supported, free and commonly deployed clinical databasing platform, REDCap. This template can be used to set up a new tuberculosis research database with a built-in set of standardised data conventions, to ensure standardised data capture across research projects and programs. A modular design enables researchers to implement only the modules of the database template that are appropriate for their particular study. The template includes core modules for informed consent data, participant demographics, clinical symptoms and presentation, diagnostic imaging and laboratory tests. Optional modules have been designed for visit scheduling and calendar functionality, clinical trial randomisation, study logistics and operations, and pharmacokinetic data. Additional fields can be added as needed. This REDCap template can facilitate collection of high-quality data for tuberculosis research, providing a tool to ensure better data harmonisation, analysis and meta-analysis.

Inflammatory profile of patients with tuberculosis with or without HIV-1 co-infection: a prospective cohort study and immunological network analysis.

BACKGROUND: HIV-1 mediated dysregulation of the immune response to tuberculosis and its effect on the response to antitubercular therapy (ATT) is incompletely understood. We aimed to analyse the inflammatory profile of patients with tuberculosis with or without HIV-1 co-infection undergoing ATT, with specific focus on the effect of ART and HIV-1 viraemia in those co-infected with HIV-1. METHODS: In this prospective cohort study and immunological network analysis, a panel of 38 inflammatory markers were measured in the plasma of a prospective patient cohort undergoing ATT at Khayelitsha Site B clinic, Cape Town, South Africa. We recruited patients with sputum Xpert MTB/RIF-positive rifampicin-susceptible pulmonary tuberculosis. Patients were excluded from the primary discovery cohort if they were younger than 18 years, unable to commence ATT for any reason, pregnant, had unknown HIV-1 status, were unable to consent to study participation, were unable to provide baseline sputum samples, had more than three doses of ATT, or were being re-treated for tuberculosis within 6 months of their previous ATT regimen. Plasma samples were collected at baseline (1-5 days after commencing ATT), week 8, and week 20 of ATT. We applied network and multivariate analysis to investigate the dynamic inflammatory profile of these patients in relation to ATT and by HIV status. In addition to the discovery cohort, a validation cohort of patients with HIV-1 admitted to hospital with CD4 counts less than 350 cells per µL and a high clinical suspicion of new tuberculosis were recruited. FINDINGS: Between March 1, 2013, and July 31, 2014, we assessed a cohort of 129 participants (55 [43%] female and 74 [57%] male, median age 35·1 years [IQR 30·1-43·7]) and 76 were co-infected with HIV-1. HIV-1 status markedly influenced the inflammatory profile regardless of ATT duration. HIV-1 viral load emerged as a major factor driving differential inflammatory marker expression and having a strong effect on correlation profiles observed in the HIV-1 co-infected group. Interleukin (IL)-17A emerged as a key correlate of HIV-1-induced inflammation during HIV-tuberculosis co-infection. INTERPRETATION: Our findings show the effect of HIV-1 co-infection on the complexity of plasma inflammatory profiles in patients with tuberculosis. Through network analysis we identified IL-17A as an important node in HIV-tuberculosis co-infection, thus implicating this cytokine's capacity to correlate with, and regulate, other inflammatory markers. Further mechanistic studies are required to identify specific IL-17A-related inflammatory pathways mediating immunopathology in HIV-tuberculosis co-infection, which could illuminate targets for future host-directed therapies. FUNDING: National Institutes of Health, The Wellcome Trust, UK Research and Innovation, Cancer Research UK, European and Developing Countries Clinical Trials Partnership, and South African Medical Research Council.

Mycobacterium tuberculosis-specific CD4 T cells expressing CD153 inversely associate with bacterial load and disease severity in human tuberculosis.

Recent data from mice and non-human primate models of tuberculosis suggested that CD153, a TNF super family member, plays an important role in Mycobacterium tuberculosis (Mtb) control. However, this molecule has not been comprehensively evaluated in humans. Here, we show that the proportion of Mtb-specific CD4 T cells expressing CD153 was significantly reduced in active TB patients compared to latently infected persons. Importantly, the CD153+ Mtb-specific CD4 response inversely correlated with lung bacterial load, inferred by Xpert cycle threshold, irrespective of HIV status. Antitubercular treatment partially restored CD153 expression on Mtb-specific CD4 T cells. This is the first report of a subset of Mtb-specific CD4 T cells showing strong negative correlation with bacterial burden. Building on substantial evidence from animal models implicating CD153 as a mediator of host protection, our findings suggest it may play a similar role in humans and its measurement may be useful to evaluate TB vaccine efficacy.

Rapid, simplified whole blood-based multiparameter assay to quantify and phenotype SARS-CoV-2 specific T cells.

Rapid tests to evaluate SARS-CoV-2-specific T cell responses are urgently needed to decipher protective immunity and aid monitoring vaccine-induced immunity. Using a rapid whole blood assay requiring minimal amount of blood, we measured qualitatively and quantitatively SARS-CoV-2-specific CD4 T cell responses in 31 healthcare workers, using flow cytometry. 100% of COVID-19 convalescent participants displayed a detectable SARS-CoV-2-specific CD4 T cell response. SARS-CoV-2-responding cells were also detected in 40.9% of participants with no COVID-19-associated symptoms or who tested PCR negative. Phenotypic assessment indicated that, in COVID-19 convalescent participants, SARS-CoV-2 CD4 responses displayed an early differentiated memory phenotype with limited capacity to produce IFNγ. Conversely, in participants with no reported symptoms, SARS-CoV-2 CD4 responses were enriched in late differentiated cells, co-expressing IFNγ and TNFα and also Granzyme B. This proof of concept study presents a scalable alternative to PBMC-based assays to enumerate and phenotype SARS-CoV-2-responding T cells, thus representing a practical tool to monitor adaptive immunity in vaccine trials. SUMMARY: In this proof of concept study, we show that SARS-CoV-2 T cell responses are easily detectable using a rapid whole blood assay requiring minimal blood volume. Such assay could represent a suitable tool to monitor adaptive immunity in vaccine trials.