MR1-restricted T cell clonotypes are associated with "resistance" to Mycobacterium tuberculosis infection.

T cells are required for protective immunity against Mycobacterium tuberculosis. We recently described a cohort of Ugandan household contacts of tuberculosis cases who appear to "resist" M. tuberculosis infection (resisters; RSTRs) and showed that these individuals harbor IFN-γ-independent T cell responses to M. tuberculosis-specific peptide antigens. However, T cells also recognize nonprotein antigens via antigen-presenting systems that are independent of genetic background, known as donor-unrestricted T cells (DURTs). We used tetramer staining and flow cytometry to characterize the association between DURTs and "resistance" to M. tuberculosis infection. Peripheral blood frequencies of most DURT subsets were comparable between RSTRs and latently infected controls (LTBIs). However, we observed a 1.65-fold increase in frequency of MR1-restricted T (MR1T) cells among RSTRs in comparison with LTBIs. Single-cell RNA sequencing of 18,251 MR1T cells sorted from 8 donors revealed 5,150 clonotypes that expressed a common transcriptional program, the majority of which were private. Sequencing of the T cell receptor α/T cell receptor δ (TCRα/δ) repertoire revealed several DURT clonotypes were expanded among RSTRs, including 2 MR1T clonotypes that recognized mycobacteria-infected cells in a TCR-dependent manner. Overall, our data reveal unexpected donor-specific diversity in the TCR repertoire of human MR1T cells as well as associations between mycobacteria-reactive MR1T clonotypes and resistance to M. tuberculosis infection.

High-parameter phenotypic characterization reveals a subset of human Th17 cells that preferentially produce IL-17 against M. tuberculosis antigen.

Background: Interleukin-17-producing CD4 T cells contribute to the control of Mycobacterium tuberculosis (Mtb) infection in humans; whether infection with human immunodeficiency virus (HIV) disproportionately affects distinct Th17-cell subsets that respond to Mtb is incompletely defined. Methods: We performed high-definition characterization of circulating Mtb-specific Th17 cells by spectral flow cytometry in people with latent TB and treated HIV (HIV-ART). We also measured kynurenine pathway activity by liquid chromatography-mass spectrometry (LC/MS) on plasma and tested the hypothesis that tryptophan catabolism influences Th17-cell frequencies in this context. Results: We identified two subsets of Th17 cells: subset 1 defined as CD4+Vα7.2-CD161+CD26+and subset 2 defined as CD4+Vα7.2-CCR6+CXCR3-cells of which subset 1 was significantly reduced in latent tuberculosis infection (LTBI) with HIV-ART, yet Mtb-responsive IL-17-producing CD4 T cells were preserved; we found that IL-17-producing CD4 T cells dominate the response to Mtb antigen but not cytomegalovirus (CMV) antigen or staphylococcal enterotoxin B (SEB), and tryptophan catabolism negatively correlates with both subset 1 and subset 2 Th17-cell frequencies. Conclusions: We found differential effects of ART-suppressed HIV on distinct subsets of Th17 cells, that IL-17-producing CD4 T cells dominate responses to Mtb but not CMV antigen or SEB, and that kynurenine pathway activity is associated with decreases of circulating Th17 cells that may contribute to tuberculosis immunity.

Evaluation of cytokine profiles related to Mycobacterium tuberculosis latent antigens using a whole-blood assay in the Philippines.

Introduction: There is no useful method to discriminate between latent tuberculosis infection (LTBI) and active pulmonary tuberculosis (PTB). This study aimed to investigate the potential of cytokine profiles to discriminate between LTBI and active PTB using whole-blood stimulation with Mycobacterium tuberculosis (MTB) antigens, including latency-associated antigens. Materials and methods: Patients with active PTB, household contacts of active PTB patients and community exposure subjects were recruited in Manila, the Philippines. Peripheral blood was collected from the participants and used for whole-blood stimulation (WBS) with either the early secretory antigenic target and the 10-kDa culture filtrate protein (ESAT-6/CFP-10), Rv3879c or latency-associated MTB antigens, including mycobacterial DNA-binding protein 1 (MDP-1), α-crystallin (Acr) and heparin-binding hemagglutinin (HBHA). Multiple cytokine concentrations were analyzed using the Bio-Plex™ multiplex cytokine assay. Results: A total of 78 participants consisting of 15 active PTB patients, 48 household contacts and 15 community exposure subjects were eligible. The MDP-1-specific IFN-γ level in the active PTB group was significantly lower than that in the household contact group (p < 0.001) and the community exposure group (p < 0.001). The Acr-specific TNF-α and IL-10 levels in the active PTB group were significantly higher than those in the household contact (TNF-α; p = 0.001, IL-10; p = 0.001) and community exposure (TNF-α; p < 0.001, IL-10; p = 0.01) groups. However, there was no significant difference in the ESAT-6/CFP-10-specific IFN-γ levels among the groups. Conclusion: The patterns of cytokine profiles induced by latency-associated MTB antigens using WBS have the potential to discriminate between LTBI and active PTB. In particular, combinations of IFN-γ and MDP-1, TNF-α and Acr, and IL-10 and Acr are promising. This study provides the first demonstration of the utility of MDP-1-specific cytokine responses in WBS.

Construction of novel multi-epitope-based diagnostic biomarker HP16118P and its application in the differential diagnosis of Mycobacterium tuberculosis latent infection.

Tuberculosis (TB) is an infectious disease that significantly threatens human health. However, the differential diagnosis of latent tuberculosis infection (LTBI) and active tuberculosis (ATB) remains a challenge for clinicians in early detection and preventive intervention. In this study, we developed a novel biomarker named HP16118P, utilizing 16 helper T lymphocyte (HTL) epitopes, 11 cytotoxic T lymphocyte (CTL) epitopes, and 8 B cell epitopes identified from 15 antigens associated with LTBI-RD using the IEDB database. We analyzed the physicochemical properties, spatial structure, and immunological characteristics of HP16118P using various tools, which indicated that it is a hydrophilic and relatively stable alkaline protein. Furthermore, HP16118P exhibited good antigenicity and immunogenicity, while being non-toxic and non-allergenic, with the potential to induce immune responses. We observed that HP16118P can stimulate the production of high levels of IFN-γ+ T lymphocytes in individuals with ATB, LTBI, and health controls. IL-5 induced by HP16118P demonstrated potential in distinguishing LTBI individuals and ATB patients (p=0.0372, AUC=0.8214, 95% CI [0.5843 to 1.000]) with a sensitivity of 100% and specificity of 71.43%. Furthermore, we incorporated the GM-CSF, IL-23, IL-5, and MCP-3 induced by HP16118P into 15 machine learning algorithms to construct a model. It was found that the Quadratic discriminant analysis model exhibited the best diagnostic performance for discriminating between LTBI and ATB, with a sensitivity of 1.00, specificity of 0.86, and accuracy of 0.93. In summary, HP16118P has demonstrated strong antigenicity and immunogenicity, with the induction of GM-CSF, IL-23, IL-5, and MCP-3, suggesting their potential for the differential diagnosis of LTBI and ATB.

Gut microbiota composition can reflect immune responses of latent tuberculosis infection in patients with poorly controlled diabetes.

BACKGROUND: Diabetes mellitus (DM) is a major risk factor for tuberculosis (TB). Evidence has linked the DM-related dysbiosis of gut microbiota to modifiable host immunity to Mycobacterium tuberculosis infection. However, the crosslinks between gut microbiota composition and immunological effects on the development of latent TB infection (LTBI) in DM patients remain uncertain. METHODS: We prospectively obtained stool, blood samples, and medical records from 130 patients with poorly-controlled DM (pDM), defined as ever having an HbA1c > 9.0% within previous 1 year. Among them, 43 had LTBI, as determined by QuantiFERON-TB Gold in-Tube assay. The differences in the taxonomic diversity of gut microbiota between LTBI and non-LTBI groups were investigated using 16S ribosomal RNA sequencing, and a predictive algorithm was established using a random forest model. Serum cytokine levels were measured to determine their correlations with gut microbiota. RESULTS: Compared with non-LTBI group, the microbiota in LTBI group displayed a similar alpha-diversity but different beta-diversity, featuring decrease of Prevotella_9, Streptococcus, and Actinomyces and increase of Bacteroides, Alistipes, and Blautia at the genus level. The accuracy was 0.872 for the LTBI prediction model using the aforementioned 6 microbiome-based biomarkers. Compared with the non-LTBI group, the LTBI group had a significantly lower serum levels of IL-17F (p = 0.025) and TNF-α (p = 0.038), which were correlated with the abundance of the aforementioned 6 taxa. CONCLUSIONS: The study results suggest that gut microbiome composition maybe associated with host immunity relevant to TB status, and gut microbial signature might be helpful for the diagnosis of LTBI.

Characterization of natural killer cells in the blood and airways of cynomolgus macaques during Mycobacterium tuberculosis infection.

BACKGROUND: Tuberculosis (TB) is caused by Mycobacterium tuberculosis (Mtb) and kills more than 1.5 million people each year. METHODS: We examine the frequency and function of NK cells in the blood and airways over the course of Mtb infection in a TB macaque model and demonstrate differences in NK marker expression between the two compartments. Flow cytometry and intracellular cytokine staining were utilized to identify NK cell subsets (expressing NKG2A, CD56, or CD16) and function (IL-10, TNF, IL-2, IFN-g, IL-17, and CD107a). RESULTS: Blood and airway NK cell frequencies were similar during infection though there were differences in subset populations between blood and airway. Increased functional (cytokine/CD107a) parameters were observed in airway NK cells during the course of infection while none were seen in the blood. CONCLUSIONS: This study suggests that NK cells in the airway may play an important role in TB host response.

Necesidad de la determinación inmunológica del Quantiferon (Interferón-gamma Relase-Assay) para el diagnóstico de la Infección Tuberculosa latente / Need for immunological determination of Quantiferon (Interferon-gamma Relase-Assay) for the diagnosis of latent tuberculous infection

Introducción: La tuberculosis es un importante problema de salud pública, primera causa de muerte en adultos contagiados de un solo agente infeccioso. Diferenciaremos enfermedad tuberculosa activa de Infección Tuberculosa Latente. El control biológico del examen inicial de salud establece si el trabajador es portador de ITL para diferenciarlo de un posible contagio posterior con motivo del trabajo. Objetivos: Objetivo general estimar la validez del Mantoux/Booster y Quantiferon como pruebas diagnósticas de la ITL. Objetivo específico definir los casos diagnosticados como ITL. Material y Métodos: Recogida de datos de las historias clínico-laborales del personal de nueva incorporación, del Área de Salud de Zamora, años 2018-2021, se importan a una base de datos, se realiza estudio descriptivo cualitativo/cuantitativo. Resultados: De los trabajadores estudiados son tuberculina positivos el 29’1%; siendo Quantiferón positivos el 10’3%. Diagnosticamos 159 casos de ITL. Conclusión: La técnica más precisa para diagnosticar la ITL es la determinación del Quantiferón. (AU)

Introduction: Tuberculosis is a major public health problem, first cause of death in adults infected with a single infectious agent. We will differentiate active tuberculosis disease from latent tuberculosis infection. The biological control of the initial health examination establishes whether the worker is a carrier of LTTI to differentiate him/her from a possible subsequent contagion at work. Objectives: General objective to estimate the validity of Mantoux/Booster and Quantiferon as diagnostic tests for LTTI. Specific objective: To define the cases diagnosed as ITL. Material and Methods: Collection of data from the clinical-work histories of newly hired personnel, from the Zamora Health Area, years 2018-2021, imported into a database, qualitative/quantitative descriptive study is performed. Results: 29.1% of the workers studied were tuberculin positive; 10.3% were Quantiferon positive. We diagnosed 159 cases of ITL. Conclusion: The most accurate technique to diagnose ITL is the determination of Quantiferon. (AU)

Serum levels of novel anti-inflammatory cytokine Interleukin-38 in diabetes patients infected with latent tuberculosis (DM-LTB-3).

IL-38 is a recently discovered, novel anti-inflammatory cytokine, which belongs to the IL-1ß family. The role played by this cytokine in diabetes-tuberculosis nexus is not known. Serum levels of IL-38, TNF-α, IL-6, and IL-1ß in Normal Glucose Tolerance (NGT) and chronic Diabetes (DM) subjects, both with and without latent tuberculosis (LTB) (n = 256) were quantified by ELISA. While, serum levels of IL-38 were significantly reduced, the levels of TNF-α, IL-6, and IL-1ß were not altered, in LTB infected diabetes patients. While no significant secretion of IL-38 was detected in the quantiferon supernatant, secretion of TNF-α, IL-6, and IL-1ß was significantly reduced in LTB infected diabetes patients. The decreased systemic levels of IL-38 and reduced in vitro secretion of other pro-inflammatory cytokines might represent a crucial pathway associated with diabetes-tuberculosis nexus.

Effect of SARS-CoV-2 seropositivity on antigen - specific cytokine and chemokine responses in latent tuberculosis.

SARS-CoV-2 and latent Mycobacterium tuberculosis infection are both highly co-prevalent in many parts of the globe. Whether exposure to SARS-CoV-2 influences the antigen specific immune responses in latent tuberculosis has not been investigated. We examined the baseline, mycobacterial antigen and mitogen induced cytokine and chemokine responses in latent tuberculosis (LTBI) individuals with or without SARS-CoV-2 seropositivity, LTBI negative individuals with SARS-CoV-2 seropositivity and healthy control (both LTBI and SARS-CoV-2 negative) individuals. Our results demonstrated that LTBI individuals with SARS-CoV-2 seropositivity (LTBI+/IgG +) were associated with increased levels of unstimulated and TB-antigen stimulated IFNγ, IL-2, TNFα, IL-17, IL-1ß, IL-6, IL-12, IL-4, CXCL1, CXCL9 and CXCL10 when compared to those without seropositivity (LTBI+/IgG-). In contrast, LTBI+/IgG+ individuals were associated with decreased levels of IL-5 and IL-10. No significant difference in the levels of cytokines/chemokines was observed upon mitogen stimulation between the groups. SARS-CoV-2 seropositivity was associated with enhanced unstimulated and TB-antigen stimulated but not mitogen stimulated production of cytokines and chemokines in LTBI+ compared to LTBI negative individuals. Finally, most of these significant differences were not observed when LTBI negative individuals with SARS-CoV-2 seropositivity and controls were examined. Our data clearly demonstrate that both baseline and TB - antigen induced cytokine responses are augmented in the presence of SARS-CoV-2 seropositivity, suggesting an augmenting effect of prior SARS-CoV-2 infection on the immune responses of LTBI individuals.

Combination of HLA-DR on Mycobacterium tuberculosis-Specific Cells and Tuberculosis Antigen/Phytohemagglutinin Ratio for Discriminating Active Tuberculosis From Latent Tuberculosis Infection.

Background: Novel approaches for tuberculosis (TB) diagnosis, especially for distinguishing active TB (ATB) from latent TB infection (LTBI), are urgently warranted. The present study aims to determine whether the combination of HLA-DR on Mycobacterium tuberculosis (MTB)-specific cells and TB antigen/phytohemagglutinin (TBAg/PHA) ratio could facilitate MTB infection status discrimination. Methods: Between June 2020 and June 2021, participants with ATB and LTBI were recruited from Tongji Hospital (Qiaokou cohort) and Sino-French New City Hospital (Caidian cohort), respectively. The detection of HLA-DR on MTB-specific cells upon TB antigen stimulation and T-SPOT assay were simultaneously performed on all subjects. Results: A total of 116 (54 ATB and 62 LTBI) and another 84 (43 ATB and 41 LTBI) cases were respectively enrolled from Qiaokou cohort and Caidian cohort. Both HLA-DR on IFN-γ+TNF-α+ cells and TBAg/PHA ratio showed discriminatory value in distinguishing between ATB and LTBI. Receiver operator characteristic (ROC) curve analysis showed that HLA-DR on IFN-γ+TNF-α+ cells produced an area under the ROC curve (AUC) of 0.886. Besides, TBAg/PHA ratio yield an AUC of 0.736. Furthermore, the combination of these two indicators resulted in the accurate discrimination with an AUC of 0.937. When the threshold was set as 0.36, the diagnostic model could differentiate ATB from LTBI with a sensitivity of 92.00% and a specificity of 81.82%. The performance obtained in Qiaokou cohort was further validated in Caidian cohort. Conclusions: The combination of HLA-DR on MTB-specific cells and TBAg/PHA ratio could serve as a robust tool to determine TB disease states.

Myeloid-Derived Suppressor Cells Mediate T Cell Dysfunction in Nonhuman Primate TB Granulomas.

Myeloid-derived suppressor cells (MDSCs) represent an innate immune cell population comprised of immature myeloid cells and myeloid progenitors with very potent immunosuppressive potential. MDSCs are reported to be abundant in the lungs of active tuberculosis (TB) patients. We sought to perform an in-depth study of MDSCs during latent TB infection (LTBI) and active TB (ATB) using the nonhuman primate (NHP) model of pulmonary TB. We found a higher proportion of granulocytic, polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs) in the lungs of ATB animals compared to those with LTBI or naive control animals. Active disease in the lung, but not LTBI, was furthermore associated with higher proliferation, expansion, and immunosuppressive capabilities of PMN-MDSCs, as shown by enhanced expression of Ki67, indoleamine 2,3-dioxygenase (IDO1), interleukin-10 (IL-10), matrix metallopeptidase 9 (MMP-9), inducible nitric oxide synthase (iNOS), and programmed death-ligand 1 (PD-L1). These immunosuppressive PMN-MDSCs specifically localized to the lymphocytic cuff at the periphery of the granulomas in animals with ATB. Conversely, these cells were scarcely distributed in interstitial lung tissue and the inner core of granulomas. This spatial regulation suggests an important immunomodulatory role of PMN-MDSCs by restricting T cell access to the TB granuloma core and can potentially explain dysfunctional anti-TB responses in active granuloma. Our results raise the possibility that the presence of MDSCs can serve as a biomarker for ATB, while their disappearance can indicate successful therapy. Furthermore, MDSCs may serve as a potential target cell for adjunctive TB therapy. IMPORTANCE Myeloid cells are immunocytes of innate origin that orchestrate the first response toward pathogens via immune surveillance (uptake and killing), antigen presentation, and initiation of adaptive immunity by T cell stimulation. However, MDSCs are a subset of innate immunocytes that deviate to an immunoregulatory phenotype. MDSCs possess strong immunosuppressive capabilities that are induced in autoimmune, malignant neoplastic, and chronic inflammatory diseases. Induction of MDSCs has been found in peripheral blood, bronchoalveolar lavage (BAL) fluid, and pleural effusions of active TB patients, but their precise localization in lung tissue and in TB granulomas remains unclear due to challenges associated with sampling lungs and granulomas from active TB patients. Nonhuman primates (NHPs) are an important animal model with TB granulomas that closely mimic those found in humans and can therefore be used for studies that are otherwise challenging with patient material. Herein, we study MDSC localization in the lungs of NHPs exhibiting latent and active TB. Our findings reveal that MDSCs localize and exert their immunosuppressive roles at the periphery rather than in the core of TB granulomas.

High Dimensional Immune Profiling Reveals Different Response Patterns in Active and Latent Tuberculosis Following Stimulation With Mycobacterial Glycolipids.

Upon infection with Mycobacterium tuberculosis (Mtb) the host immune response might clear the bacteria, control its growth leading to latent tuberculosis (LTB), or fail to control its growth resulting in active TB (ATB). There is however no clear understanding of the features underlying a more or less effective response. Mtb glycolipids are abundant in the bacterial cell envelope and modulate the immune response to Mtb, but the patterns of response to glycolipids are still underexplored. To identify the CD45+ leukocyte activation landscape induced by Mtb glycolipids in peripheral blood of ATB and LTB, we performed a detailed assessment of the immune response of PBMCs to the Mtb glycolipids lipoarabinomannan (LAM) and its biosynthetic precursor phosphatidyl-inositol mannoside (PIM), and purified-protein derivate (PPD). At 24 h of stimulation, cell profiling and secretome analysis was done using mass cytometry and high-multiplex immunoassay. PIM induced a diverse cytokine response, mainly affecting antigen-presenting cells to produce both pro-inflammatory and anti-inflammatory cytokines, but not IFN-γ, contrasting with PPD that was a strong inducer of IFN-γ. The effect of PIM on the antigen-presenting cells was partly TLR2-dependent. Expansion of monocyte subsets in response to PIM or LAM was reduced primarily in LTB as compared to healthy controls, suggesting a hyporesponsive/tolerance pattern derived from Mtb infection.

A differential DNA methylome signature of pulmonary immune cells from individuals converting to latent tuberculosis infection.

Tuberculosis (TB), caused by Mycobacterium tuberculosis, spreads via aerosols and the first encounter with the immune system is with the pulmonary-resident immune cells. The role of epigenetic regulations in the immune cells is emerging and we have previously shown that macrophages capacity to kill M. tuberculosis is reflected in the DNA methylome. The aim of this study was to investigate epigenetic modifications in alveolar macrophages and T cells in a cohort of medical students with an increased risk of TB exposure, longitudinally. DNA methylome analysis revealed that a unique DNA methylation profile was present in healthy subjects who later developed latent TB during the study. The profile was reflected in a different overall DNA methylation distribution as well as a distinct set of differentially methylated genes (DMGs). The DMGs were over-represented in pathways related to metabolic reprogramming of macrophages and T cell migration and IFN-γ production, pathways previously reported important in TB control. In conclusion, we identified a unique DNA methylation signature in individuals, with no peripheral immune response to M. tuberculosis antigen who later developed latent TB. Together the study suggests that the DNA methylation status of pulmonary immune cells can reveal who will develop latent TB infection.

Serum Biomarker Profile Including CCL1, CXCL10, VEGF, and Adenosine Deaminase Activity Distinguishes Active From Remotely Acquired Latent Tuberculosis.

Introduction: There is an urgent medical need to differentiate active tuberculosis (ATB) from latent tuberculosis infection (LTBI) and prevent undertreatment and overtreatment. The aim of this study was to identify biomarker profiles that may support the differentiation between ATB and LTBI and to validate these signatures. Materials and Methods: The discovery cohort included adult individuals classified in four groups: ATB (n = 20), LTBI without prophylaxis (untreated LTBI; n = 20), LTBI after completion of prophylaxis (treated LTBI; n = 20), and healthy controls (HC; n = 20). Their sera were analyzed for 40 cytokines/chemokines and activity of adenosine deaminase (ADA) isozymes. A prediction model was designed to differentiate ATB from untreated LTBI using sparse partial least squares (sPLS) and logistic regression analyses. Serum samples of two independent cohorts (national and international) were used for validation. Results: sPLS regression analyses identified C-C motif chemokine ligand 1 (CCL1), C-reactive protein (CRP), C-X-C motif chemokine ligand 10 (CXCL10), and vascular endothelial growth factor (VEGF) as the most discriminating biomarkers. These markers and ADA(2) activity were significantly increased in ATB compared to untreated LTBI (p ≤ 0.007). Combining CCL1, CXCL10, VEGF, and ADA2 activity yielded a sensitivity and specificity of 95% and 90%, respectively, in differentiating ATB from untreated LTBI. These findings were confirmed in the validation cohort including remotely acquired untreated LTBI participants. Conclusion: The biomarker signature of CCL1, CXCL10, VEGF, and ADA2 activity provides a promising tool for differentiating patients with ATB from non-treated LTBI individuals.

Identification of Hub Genes in Tuberculosis via Bioinformatics Analysis.

BACKGROUND: Tuberculosis (TB) is a serious chronic bacterial infection caused by Mycobacterium tuberculosis (MTB). It is one of the deadliest diseases in the world and a heavy burden for people all over the world. However, the hub genes involved in the host response remain largely unclear. METHODS: The data set GSE11199 was studied to clarify the potential gene network and signal transduction pathway in TB. The subjects were divided into latent tuberculosis and pulmonary tuberculosis, and the distribution of differentially expressed genes (DEGs) was analyzed between them using GEO2R. We verified the enriched process and pathway of DEGs by making use of the Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO). The construction of protein-protein interaction (PPI) network of DEGs was achieved through making use of the Search Tool for the Retrieval of Interacting Genes (STRING), aiming at identifying hub genes. Then, the hub gene expression level in latent and pulmonary tuberculosis was verified by a boxplot. Finally, through making use of Gene Set Enrichment Analysis (GSEA), we further analyzed the pathways related to DEGs in the data set GSE11199 to show the changing pattern between latent and pulmonary tuberculosis. RESULTS: We identified 98 DEGs in total in the data set GSE11199, 91 genes upregulated and 7 genes downregulated included. The enrichment of GO and KEGG pathways demonstrated that upregulated DEGs were mainly abundant in cytokine-mediated signaling pathway, response to interferon-gamma, endoplasmic reticulum lumen, beta-galactosidase activity, measles, JAK-STAT signaling pathway, cytokine-cytokine receptor interaction, etc. Based on the PPI network, we obtained 4 hub genes with a higher degree, namely, CTLA4, GZMB, GZMA, and PRF1. The box plot showed that these 4 hub gene expression levels in the pulmonary tuberculosis group were higher than those in the latent group. Finally, through Gene Set Enrichment Analysis (GSEA), it was concluded that DEGs were largely associated with proteasome and primary immunodeficiency. CONCLUSIONS: This study reveals the coordination of pathogenic genes during TB infection and offers the diagnosis of TB a promising genome. These hub genes also provide new directions for the development of latent molecular targets for TB treatment.

Type 2 Diabetes Mellitus and Latent Tuberculosis Infection Moderately Influence Innate Lymphoid Cell Immune Responses in Uganda.

Background: Type 2 diabetes mellitus (T2DM) is a major risk factor for the acquisition of latent tuberculosis (TB) infection (LTBI) and development of active tuberculosis (ATB), although the immunological basis for this susceptibility remains poorly characterised. Innate lymphoid cells (ILCs) immune responses to TB infection in T2DM comorbidity is anticipated to be reduced. We compared ILC responses (frequency and cytokine production) among adult patients with LTBI and T2DM to patients (13) with LTBI only (14), T2DM only (10) and healthy controls (11). Methods: Using flow cytometry, ILC phenotypes were categorised based on (Lin-CD127+CD161+) markers into three types: ILC1 (Lin-CD127+CD161+CRTH2-CD117-); ILC2 (Lin-CD127+CD161+CRTH2+) and ILC3 (Lin-CD127+CD161+CRTH2-NKp44+/-CD117+). ILC responses were determined using cytokine production by measuring percentage expression of interferon-gamma (IFN-γ) for ILC1, interleukin (IL)-13 for ILC2, and IL-22 for ILC3. Glycaemic control among T2DM patients was measured using glycated haemoglobin (HbA1c) levels. Data were analysed using FlowJo version 10.7.1, and GraphPad Prism version 8.3. Results: Compared to healthy controls, patients with LTBI and T2DM had reduced frequencies of ILC2 and ILC3 respectively (median (IQR): 0.01 (0.005-0.04) and 0.002 (IQR; 0.002-0.007) and not ILC1 (0.04 (0.02-0.09) as expected. They also had increased production of IFN-γ [median (IQR): 17.1 (5.6-24.9)], but decreased production of IL-13 [19.6 (12.3-35.1)]. We however found that patients with T2DM had lower ILC cytokine responses in general but more marked for IL-22 production (median (IQR): IFN-γ 9.3 (4.8-22.6); IL-13 22.2 (14.7-39.7); IL-22 0.7 (IQR; 0.1-2.1) p-value 0.02), which highlights the immune suppression status of T2DM. We also found that poor glycaemic control altered ILC immune responses. Conclusion: This study demonstrates that LTBI and T2DM, and T2DM were associated with slight alterations of ILC immune responses. Poor T2DM control also slightly altered these ILC immune responses. Further studies are required to assess if these responses recover after treatment of either TB or T2DM.

Activation Phenotype of Mycobacterium tuberculosis-Specific CD4+ T Cells Promoting the Discrimination Between Active Tuberculosis and Latent Tuberculosis Infection.

Background: Rapid and effective discrimination between active tuberculosis (ATB) and latent tuberculosis infection (LTBI) remains a challenge. There is an urgent need for developing practical and affordable approaches targeting this issue. Methods: Participants with ATB and LTBI were recruited at Tongji Hospital (Qiaokou cohort) and Sino-French New City Hospital (Caidian cohort) based on positive T-SPOT results from June 2020 to January 2021. The expression of activation markers including HLA-DR, CD38, CD69, and CD25 was examined on Mycobacterium tuberculosis (MTB)-specific CD4+ T cells defined by IFN-γ, TNF-α, and IL-2 expression upon MTB antigen stimulation. Results: A total of 90 (40 ATB and 50 LTBI) and another 64 (29 ATB and 35 LTBI) subjects were recruited from the Qiaokou cohort and Caidian cohort, respectively. The expression patterns of Th1 cytokines including IFN-γ, TNF-α, and IL-2 upon MTB antigen stimulation could not differentiate ATB patients from LTBI individuals well. However, both HLA-DR and CD38 on MTB-specific cells showed discriminatory value in distinguishing between ATB patients and LTBI individuals. As for developing a single candidate biomarker, HLA-DR had the advantage over CD38. Moreover, HLA-DR on TNF-α+ or IL-2+ cells had superiority over that on IFN-γ+ cells in differentiating ATB patients from LTBI individuals. Besides, HLA-DR on MTB-specific cells defined by multiple cytokine co-expression had a higher ability to discriminate patients with ATB from LTBI individuals than that of MTB-specific cells defined by one kind of cytokine expression. Specially, HLA-DR on TNF-α+IL-2+ cells produced an AUC of 0.901 (95% CI, 0.833-0.969), with a sensitivity of 93.75% (95% CI, 79.85-98.27%) and specificity of 72.97% (95% CI, 57.02-84.60%) as a threshold of 44% was used. Furthermore, the performance of HLA-DR on TNF-α+IL-2+ cells for differential diagnosis was obtained with validation cohort data: 90.91% (95% CI, 72.19-97.47%) sensitivity and 68.97% (95% CI, 50.77-82.73%) specificity. Conclusions: We demonstrated that HLA-DR on MTB-specific cells was a potentially useful biomarker for accurate discrimination between ATB and LTBI.

Non-IFNγ Whole Blood Cytokine Responses to Mycobacterium tuberculosis Antigens in HIV-exposed Infants.

BACKGROUND: HIV-exposed uninfected (HEU) infants have increased risk of tuberculosis (TB). Testing for Mycobacterium tuberculosis (Mtb) infection is limited by reduced Quantiferon (QFT) sensitivity in infants and tuberculin skin test (TST) cross-reactivity with Bacillus Calmette-Guérin vaccine. Our objective is to assess if non-IFNγ cytokine responses to Mtb-specific antigens have improved sensitivity in detecting Mtb infection in HEU infants compared with QFT. METHODS: HEU infants were enrolled in a randomized clinical trial of isoniazid preventive therapy (IPT) to prevent Mtb infection in Kenya (N = 300) and assessed at 12 months postrandomization (14 months of age) by TST and QFT-Plus. Non-IFNγ cytokine secretion (IL2, TNF, IP10, N = 229) in QFT-Plus supernatants was measured using Luminex assay. Logistic regression was used to assess the effect of IPT on Mtb infection outcomes in HEU infants. RESULTS: Three of 251 (1.2%) infants were QFT-Plus positive. Non-IFNγ Mtb antigen-specific responses were detected in 12 additional infants (12/229, 5.2%), all TST negative. IPT was not associated with Mtb infection defined as any Mtb antigen-specific cytokine response (odds ratio = 0.7, P = 0.54). Mtb antigen-specific IL2/IP10 responses had fair correlation (τ = 0.25). Otherwise, non-IFNγ cytokine responses had minimal correlation with QFT-Plus and no correlation with TST size. CONCLUSIONS: We detected non-IFNg Mtb antigen-specific T-cell responses in 14-month HEU infants. Non-IFNg cytokines may be more sensitive than IFNg in detecting infant Mtb infection. IPT during the first year of life was not associated with Mtb infection measured by IFNg, IL2, IP10 and TNF Mtb-specific responses.

Defective expansion and function of memory like natural killer cells in HIV+ individuals with latent tuberculosis infection.

PURPOSE: Tuberculosis (TB) is the leading cause of infectious disease related mortality, and only 10% of the infected individuals develop active disease. The likelihood of progression of latent tuberculosis infection (LTBI) to active TB disease is high in HIV infected individuals. Identification of HIV+ individuals at risk would allow treating targeted population, facilitating completion of therapy for LTBI and prevention of TB development. NK cells have an important role in T cell independent immunity against TB, but the exact role of NK cell subsets in LTBI and HIV is not well characterized. METHODS: In this study, we compared the expansion and function of memory like NK cells from HIV-LTBI+ individuals and treatment naïve HIV+LTBI+ patients in response to Mtb antigens ESAT-6 and CFP-10. RESULTS: In freshly isolated PBMCs, percentages of CD3-CD56+ NK cells were similar in HIV+LTBI+ patients and healthy HIV-LTBI+ individuals. However, percentages of CD3-CD56+CD16+ NK cells were higher in healthy HIV-LTBI+ individuals compared to HIV+LTBI+ patients. HIV infection also inhibited the expansion of memory like NK cells, production of IL-32α, IL-15 and IFN-γ in response to Mtb antigens in LTBI+ individuals. CONCLUSION: We studied phenotypic, functional subsets and activation of memory like-NK cells during HIV infection and LTBI. We observed that HIV+LTBI+ patients demonstrated suboptimal NK cell and monocyte interactions in response to Mtb, leading to reduced IL-15, IFN-γ and granzyme B and increased CCL5 production. Our study highlights the effect of HIV and LTBI on modulation of NK cell activity to understand their role in development of interventions to prevent progression to TB in high risk individuals.

Analysis of multivariate longitudinal immuno-epidemiological data using a pairwise joint modelling approach.

BACKGROUND: Immuno-epidemiologists are often faced with multivariate outcomes, measured repeatedly over time. Such data are characterised by complex inter- and intra-outcome relationships which must be accounted for during analysis. Scientific questions of interest might include determining the effect of a treatment on the evolution of all outcomes together, or grouping outcomes that change in the same way. Modelling the different outcomes separately may not be appropriate because it ignores the underlying relationships between outcomes. In such situations, a joint modelling strategy is necessary. This paper describes a pairwise joint modelling approach and discusses its benefits over more simple statistical analysis approaches, with application to data from a study of the response to BCG vaccination in the first year of life, conducted in Entebbe, Uganda. METHODS: The study aimed to determine the effect of maternal latent Mycobacterium tuberculosis infection (LTBI) on infant immune response (TNF, IFN-γ, IL-13, IL-10, IL-5, IL-17A and IL-2 responses to PPD), following immunisation with BCG. A simple analysis ignoring the correlation structure of multivariate longitudinal data is first shown. Univariate linear mixed models are then used to describe longitudinal profiles of each outcome, and are then combined into a multivariate mixed model, specifying a joint distribution for the random effects to account for correlations between the multiple outcomes. A pairwise joint modelling approach, where all possible pairs of bivariate mixed models are fitted, is then used to obtain parameter estimates. RESULTS: Univariate and pairwise longitudinal analysis approaches are consistent in finding that LTBI had no impact on the evolution of cytokine responses to PPD. Estimates from the pairwise joint modelling approach were more precise. Major advantages of the pairwise approach include the opportunity to test for the effect of LTBI on the joint evolution of all, or groups of, outcomes and the ability to estimate association structures of the outcomes. CONCLUSIONS: The pairwise joint modelling approach reduces the complexity of analysis of high-dimensional multivariate repeated measures, allows for proper accounting for association structures and can improve our understanding and interpretation of longitudinal immuno-epidemiological data.