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Background:Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), is a persistent infectious disease threatening human health. The existing diagnostic methods still have significant shortcomings, including a low positivity rate in pathogen-based diagnoses and the inability of immunological diagnostics to detect active TB. Hence, it is urgent to develop new techniques to detect TB more accurate and earlier. This research aims to scrutinize and authenticate DNA methylation markers suitable for tuberculosis diagnosis. Concurrently, Providing a new approach for tuberculosis diagnosis.Methods:Blood samples from patients with newly diagnosed tuberculosis and healthy controls (HC) were utilized in this study. Examining methylation microarray data from 40 whole blood samples (22TB + 18HC), we employed two procedures: signature gene methylated position analysis and signature region methylated position analysis to pinpoint distinctive methylated positions. Based on the screening results, diagnostic classifiers are constructed through machine learning, and validation was conducted through pyrosequencing in a separate queue (22TB + 18HC). Culminating in the development of a new tuberculosis diagnostic method via quantitative real-time methylation specific PCR (qMSP).Results:The combination of the two procedures revealed a total of 10 methylated positions, all of which were located in the promoter region. These 10 signature methylated positions facilitated the construction of a diagnostic classifier, exhibiting robust diagnostic accuracy in both cross-validation and external test sets. The LDA model demonstrated the best classification performance, achieving an AUC of 0.83, specificity of 0.8, and sensitivity of 0.86 on the external test set. Furthermore, the validation of signature methylated positions through pyrosequencing demonstrated high agreement with screening outcomes. Additionally, qMSP detection of 2 potential hypomethylated positions (cg04552852 and cg12464638) exhibited promising results, yielding an AUC of 0.794, specificity of 0.720, and sensitivity of 0.816.Conclusion:Our study demonstrates that the validated signature methylated positions through pyrosequencing emerge as plausible biomarkers for tuberculosis diagnosis. The specific methylation markers in the TSPAN4 gene, identified in whole blood samples, hold promise for improving tuberculosis diagnosis. This approach could significantly enhance diagnostic accuracy and speed, offering a new avenue for early detection and treatment.

Abstract Introduction The urgent need for new treatments for multidrug-resistant tuberculosis (MDR-TB) and pre-extensively drug-resistant tuberculosis (pre-XDR-TB) is evident. However, the classic randomized controlled trial (RCT) approach faces ethical and practical constraints, making alternative research designs and treatment strategies necessary, such as single-arm trials and host-directed therapies (HDTs). Methods Our study adopts a randomized withdrawal trial design for MDR-TB to maximize resource allocation and better mimic real-world conditions. Patients’ treatment regimens are initially based on drug resistance profiles and patient’s preference, and later, treatment-responsive cases are randomized to different treatment durations. Alongside, a single-arm trial is being conducted to evaluate the potential of sulfasalazine (SASP) as an HDT for pre-XDR-TB, as well as another short-course regimen without HDT for pre-XDR-TB. Both approaches account for the limitations in second-line anti-TB drug resistance testing in various regions. Discussion Although our study designs may lack the internal validity commonly associated with RCTs, they offer advantages in external validity, feasibility, and ethical appropriateness. These designs align with real-world clinical settings and also open doors for exploring alternative treatments like SASP for tackling drug-resistant TB forms. Ultimately, our research aims to strike a balance between scientific rigor and practical utility, offering valuable insights into treating MDR-TB and pre-XDR-TB in a challenging global health landscape. In summary, our study employs innovative trial designs and treatment strategies to address the complexities of treating drug-resistant TB, fulfilling a critical gap between ideal clinical trials and the reality of constrained resources and ethical considerations. Trail registration Chictr.org.cn, ChiCTR2100045930. Registered on April 29, 2021.

Objectives: Long, ineffective, and toxic regimens hinder the treatment of patients with multidrug-resistant tuberculosis (MDR-TB) and pre-extensive drug-resistant tuberculosis (pre-XDR-TB). Methods: We conducted a multicenter cohort study to prospectively evaluate the safety and efficacy of three 9-month, all-oral, 5-drug regimens. Regimen A (bedaquiline [Bdq]+linezolid [Lzd]+moxifloxacin [Mfx]+cycloserine [Cs]+pyrazinamide [Pza]) and Regimen B (Lzd+Mfx+Cs+clofazimine [Cfz]+Pza) were used to treat MDR-TB patients (Groups A and B, respectively, assigned according to the patient's treatment preference), while Regimen C (Bdq+Lzd+Cs+Cfz+Pza) was used to treat pre-XDR-TB patients (Group C). The primary endpoint was the occurrence of an unfavorable outcome within 12 months of treatment completion, regardless of regimen. Results: A total of 104 patients (34 in Group A, 46 in Group B, and 24 in Group C), with a median age of 35.5 (29.0-54.0) years, were included in the analysis population. At 12 months after treatment completion, five patients were deemed non-assessable. Of the remaining 99 participants, seven (7.1%) had an unfavorable outcome (including two deaths from any cause, four with treatment failure, and one loss to follow-up) and 92 (92.9%) had a favorable outcome. Culture conversion was achieved in 82.5% (80/97) of participants at month 2 and in 97.9% (94/97) of participants at month 6. Adverse events (AEs) resulting in drug adjustment occurred in 69.2% (72/104) of participants, mainly due to Lzd and Pza use. A QT interval prolongation of ≥ 500 ms occurred in 5.8% (6/104) of participants. Conclusion: The primary outcome of the three tailored, 9-month, all-oral, 5-drug regimens was satisfactory in the vast majority of MDR-TB and pre-XDR-TB patients, with manageable and reversible AEs.

Mycobacterium tuberculosis (Mtb) infection, including active tuberculosis (TB) and latent Mtb infection (LTBI), leads to diverse outcomes owing to different host immune responses. However, the immune mechanisms that govern the progression from LTBI to TB remain poorly defined in humans. Here, we profiled the lung immune cell populations within the bronchoalveolar lavage fluid (BALF) from patients with LTBI or TB using single-cell RNA sequencing (scRNA-seq). We found that Mtb infection substantially changed the immune cell compartments in the BALF, especially for the three subsets of macrophages, monocyte macrophage (MM)-CCL23, MM-FCN1, and MM-SPP1, which were found to be associated with the disease status of TB infection. Notably, MM-CCL23 cells derived from monocytes after stimulation with Mtb were characterized by high levels of chemokine (CCL23 and CXCL5) production and might serve as a marker for Mtb infection. The MM-CCL23 population mainly recruited CD8-CCR6 T cells through CCL20/CCR6, which was a prominent feature associated with protection immunity in LTBI. This study improves our understanding of the lung immune landscape during Mtb infection, which may inform future vaccine design for protective immunity.

Key messages What is already known on this topic—Breath VOC analysis is a potential technology for PTB detection. However, it is still desirable for a real-time, robust, accurate, and simple breath analysis platform for clinical application. What this study adds—An online breath detection for PTB was proposed and demonstrated with high sensitivity and specificity in a large clinical cohort. How this study might affect research, practice, or policy—This study may promote the application of breath detection in clinical TB detection and related biomarker studies.

Objectives: Increased resistance of Candida species, especially Candida. glabrata is problematic. Combination antifungal therapies were studied to solve the problem. Methods: In this study, combinations of caspofungin with fluconazole and voriconazole were evaluated in 28 Candida species (including 15 C. glabrata and 12 with FKS mutation) at 24 and 48 hours using two Etest methods (direct cover method and MIC/MIC method). Results: For Candida isolates, direct cover method showed synergy of caspofungin-fluconazole and caspofungin-voriconazole against 12/28 (43%) isolates at 24 hours, and against 16/28 (57%) isolates at 48 hours. The MIC/MIC method showed synergy of caspofungin-fluconazole and caspofungin-voriconazole against 11/28 (39%) and 12/28 (43%) isolates at 24 hours, and against 16/28 (57%) and 17/28 (61%) isolates at 48 hours, respectively. For C. glabrata, direct cover method showed synergy of caspofungin-fluconazole and caspofungin-voriconazole against 11/15 (73%) and 10/15 (67%) isolates at 24 hours, and 11/15 (73%) and 13/15 (87%) isolates at 48 hours, respectively. The MIC/MIC method showed synergy of caspofungin-fluconazole and caspofungin-voriconazole against both 11/15 (73%) isolates at 24 hours, and 10/15 (67%) and 14/15 (93%) isolates at 48 hours, respectively. Conclusion: A combination of caspofungin and fluconazole or voriconazole might be effective against infections caused by Candida species, especially C. glabrata with FKS mutation.

Background: Multidrug-resistant tuberculosis (MDR-TB) patients have been suffering long, ineffective, and toxic treatment until short-course injectable-free regimens emerged. However, the new WHO-recommended regimens might be less feasible in the real-world setting. Here, we evaluated two optimized all-oral short-course regimens in China. Methods: From April 2019 to August 2020, we conducted a prospective nonrandomized controlled trial and consecutively included 103 MDR-TB patients diagnosed with pulmonary MDR-TB in Shenzhen, China. A 4-5 drug regimen of 9-12 months was tailored to the strain's resistance patterns, patients' affordability, and tolerance to drugs. This was an interim analysis, focusing on the early treatment period. Results: 53.4% (55/103) of patients were prescribed linezolid, fluoroquinolone (FQ), clofazimine, cycloserine, and pyrazinamide, followed by a regimen in which clofazimine was replaced by bedaquiline (35/103, 34.0%). The culture conversion rate was 83.1% and 94.4% at two and four months, respectively, with no significant difference between bedaquiline-free and bedaquiline-containing cases and between FQ-susceptible and FQ-resistant cases. Among 41 patients who completed treatment, 40 (97.6%) patients had a favorable outcome and no relapse was observed. Peripheral neuropathy and arthralgia/myalgia were the most frequent AEs (56.3%, 58/103). 18 AEs caused permanent discontinuation of drugs, mostly due to pyrazinamide and linezolid. Conclusion: Optimized all-oral short-course regimens showed satisfactory efficacy and safety in early treatment stage. Further research is needed to confirm these results.

Background: To develop a population pharmacokinetic (PPK) model for caspofungin, identify parameters influencing caspofungin pharmacokinetics, and assess the required probability of target attainment (PTA) and cumulative fraction of response (CFR) for various dosing regimens of caspofungin in all patients and intensive care unit (ICU)-subgroup patients.Method: The general PPK model was developed based on data sets from all patients (299 patients). A ICU-subgroup PPK model based on data sets from 136 patients was then analyzed. The effects of demographics, clinical data, laboratory data, and concomitant medications were tested. Monte-Carlo simulations (MCS) were used to evaluate the effectiveness of different caspofungin dosage regimens.Results: One-compartment model best described the data of all patients and ICU patients. Clearances (CL) were 0.32 L/h and 0.40 L/h and volumes of distribution (V) were 13.31 L and 10.20 L for the general and ICU-subgroup PPK models, respectively. In the general model, CL and V were significantly associated with albumin (ALB) concentration and body weight (WT). In the ICU-subgroup model, CL was associated with WT. The simulated exposure in ICU patients was lower than that in all patients (p < 0.05). MCS indicated that higher caspofungin maintenance doses of 70–150 mg may achieve target CFR of >90% for patients with higher WT (>70 kg) or with C. albicans or C. parapsilosis infections, and especially for ICU patients with hypoalbuminaemia.Conclusion: The PPK model and MCS presented in the study demonstrated that the recommended dosage regimen for caspofungin in patients with higher body weight or hypoalbuminaemia will result in low exposure.

Neutrophils have been recognized to play an important role in the pathogenesis of tuberculosis in recent years. Interferon-induced blood transcriptional signatures in ATB are predominantly driven by neutrophils. In this study, we performed global RNA-seq on peripheral blood neutrophils from active tuberculosis patients (ATB, n=15); latent tuberculosis infections (LTBI, n=22); and healthy controls (HC, n=21). The results showed that greater perturbations of gene expression patterns happened in neutrophils from ATB individuals than HC or those with LTBI, and a total of 344 differentially expressed genes (DEGs) were observed. Functional enrichment analysis showed that besides the interferon signaling pathway, multiple pattern recognition receptor pathways were significantly activated in ATB, such as NOD-like receptors and Toll-like receptors. Meanwhile, we also observed that the expression of genes related to endocytosis, secretory granules, and neutrophils degranulation were downregulated. Our data also showed that the NF-κB signaling pathway might be inhibited in patients with ATB, which could increase Mycobacterium tuberculosis survival and lead to active tuberculosis status. Furthermore, we validated the accuracy of some differentially expressed genes in an independent cohort using quantitative PCR, and obtained three novel genes (RBM3, CSRNP1, SRSF5) with the ability to discriminate active tuberculosis from LTBI and HC.

Background: Oral administration increases the risk of interactions, because most oral antineoplastic agents (OAAs) are taken on a daily basis. Interactions can increase exposure to antitumoral agents or cause treatment failure. Potential drug–drug interactions (DDIs) are commonly observed in patients with cancer, while the extent to which OAAs related hazardous DDIs remains unclear.Methods: We studied the contraindication patterns between oral antineoplastic agents and other medications among cancer patients in two tertiary care teaching hospitals in China. A total of 20 clinically significant hazardous DDI pairs that involved 30 OAAs were identified based on the predetermined criteria. Patient medications were checked for DDIs by using the US Food and Drug Administration approved labeling. Descriptive statistics and uni- and multivariate logistic regression analyses were carried out.Results: In this study, 13,917 patients were included and a total of 297 DDIs were identified. The results revealed that proton pump inhibitors (PPIs), dexamethasone and fluoroquinolones were the most often involved hazardous DDIs with OAAs. The most prevalent contraindication is the simultaneous use of certain molecular targeted agents and PPIs. In the result of the multivariate analysis, younger age (0–20 group), increasing number of drugs and patient treated with targeted therapy had a higher risk for DDIs.Conclusion: The prevalence of OAAs related hazardous DDIs appears to be low in the cancer patients. However, physicians and clinical pharmacologists should be aware of the potential hazardous DDIs when prescribing OAAs, especially certain pH-dependent molecular targeted agents and potential QTc prolonging drugs.

Summary: The worldwide epidemic of coronavirus disease 2019 (COVID-19) is ongoing. Rapid and accurate detection of the causative virus SARS-CoV-2 is vital for the treatment and control of COVID-19. In this study, the comparative sensitivity of different respiratory specimen types were retrospectively analyzed using 3,552 clinical samples from 410 COVID-19 patients confirmed by Guangdong CDC (Center for Disease Control and Prevention). Except for bronchoalveolar lavage fluid (BALF), the sputum possessed the highest positive rate (73.4%–87.5%), followed by nasal swabs (53.1%–85.3%) for both severe and mild cases during the first 14 days after illness onset (d.a.o.). Viral RNA could be detected in all BALF samples collected from the severe group within 14 d.a.o. and lasted up to 46 d.a.o. Moreover, although viral RNA was negative in the upper respiratory samples, it was also positive in BALF samples in most cases from the severe group during treatment. Notably, no viral RNA was detected in BALF samples from the mild group. Despite typical ground-glass opacity observed via computed tomographic scans, no viral RNA was detected in the first three or all upper respiratory tract specimens from some COVID-19 patients. In conclusion, sputum is most sensitive for routine laboratory diagnosis of COVID-19, followed by nasal swabs. Detection of viral RNA in BALF improves diagnostic accuracy in severe COVID-19 patients.

ABSTRACT xCT forms part of the xc− cysteine-glutamate antiporter which inhibits antimicrobial inflammatory immune functions and thus increases susceptibility to tuberculosis (TB). However, the associations between xCT gene polymorphisms and susceptibility to TB, as well as whether these modulate xCT expression or affect treatment with the xCT inhibitor sulfasalazine (SASP), are unclear. In the present study, we genotyped xCT polymorphisms in a large Chinese cohort and found that the single-nucleotide polymorphism (SNP) rs13120371 was associated with susceptibility to TB. The rs13120371 AA genotype was strongly associated with an increased risk of TB and increased xCT mRNA expression levels compared to those with the GG or AG genotype. rs13120371 is located on the 3′ untranslated (UTR) region of the xCT gene, in the putative binding site for miR-142-3p, and the results of luciferase reporter assays indicated that the rs13120371 AA genotype inhibited the binding of miR-42-3p to xCT. Bacterial burden was also significantly higher in cells with the AA genotype than in those with the GG genotype. Furthermore, pretreatment with SASP alleviated this burden in cells with the AA genotype but conferred no benefit in cells with the GG phenotype. In summary, we identified a functional SNP (rs13120371) in the xCT 3′ UTR region that increases susceptibility to TB through interacting with miR-142-3p. IMPORTANCE Tuberculosis (TB) is the leading cause of death from a single infectious agent globally, and the development of multidrug resistance represents a serious health concern, particularly in the developing world. Novel effective treatments are urgently required. xCT expression is known to increase susceptibility to TB, and certain polymorphisms in the gene encoding this protein interrupt the binding of microRNA and prevent its suppression. Taking advantage of the FDA approval for the use of sulfasalazine (SASP), which inhibits xCT-mediated cystine transport in humans, we demonstrate how host genotype-specific therapies tailored to the xCT genotype can improve TB outcomes.

Abstract Background Invasive pulmonary aspergillosis (IPA) is a life-threatening disease in immunosuppressed patients. Voriconazole is commonly used to prevent and treat IPA in the clinic, but the optimal prophylactic antifungal regimen is unknown. The objective of this study was to clarify the mechanism underlying how voriconazole prevents IPA based on a target cellular pharmacokinetics/pharmacodynamics model, with the aim of identifying a way to design an optimal prophylactic antifungal regimen. Methods A nystatin assay was used to establish a target-cells model for A. fumigatus infection. An inhibitory effect sigmoid Emax model was developed to explore the cellular PK/PD breakpoint, and Monte Carlo simulation was used to design the prophylactic antifungal regimen. Results The intracellular activity of voriconazole in the target cells varied with its concentration, with the minimum inhibitory concentration (MIC) being an important determinant. For A. fumigatus strains AF293 and AF26, voriconazole decreased the intracellular inoculum by 0.79 and 0.84 lg cfu, respectively. The inhibitory effect sigmoid Emax model showed that 84.01% of the intracellular inoculum was suppressed by voriconazole within 24 h, and that a PK/PD value of 35.53 for the extracellular voriconazole concentration divided by MIC was associated with a 50% suppression of intracellular A. fumigatus. The Monte Carlo simulation results showed that the oral administration of at least 200 mg of voriconazole twice daily was yielded estimated the cumulative fraction of response value of 91.48%. Concentration of voriconazole in the pulmonary epithelial lining fluid and the plasma of > 17.77 and > 1.55 mg/L, respectively, would ensure the PK/PD > 35.53 for voriconazole against most isolates of A. fumigatus and may will be benefit to prevent IPA in clinical applications. Conclusions This study used a target cellular pharmacokinetics/pharmacodynamics model to reveal a potential mechanism underlying how voriconazole prevents IPA and has provided a method for designing voriconazole prophylactic antifungal regimen in immunosuppressed patients.

Summary: T cells are strongly regulated by oxidizing environments and amino acid restriction. How T cells reprogram metabolism to adapt to these extracellular stress situations is not well understood. Here, we show that oxidizing environments and amino acid starvation induce ATF4 in CD4+ T cells. We also demonstrate that Atf4-deficient CD4+ T cells have defects in redox homeostasis, proliferation, differentiation, and cytokine production. We further reveal that ATF4 regulates a coordinated gene network that drives amino acid intake, mTORC1 activation, protein translation, and an anabolic program for de novo synthesis of amino acids and glutathione. ATF4 also promotes catabolic glycolysis and glutaminolysis and oxidative phosphorylation and thereby provides precursors and energy for anabolic pathways. ATF4-deficient mice mount reduced Th1 but elevated Th17 immune responses and develop more severe experimental allergic encephalomyelitis (EAE). Our study demonstrates that ATF4 is critical for CD4+ T cell-mediated immune responses through driving metabolic adaptation. : Oxidizing environments and availability of extracellular amino acids are major mechanisms that regulate T cell proliferation and function. Yang et al. demonstrate that ATF4 drives metabolic reprogramming, which allows CD4+ T cells to adapt to these stresses. Keywords: T cell, metabolism, EAE, Th17, Th1, ATF4, amino acid, glycolysis, TCA cycle, autoimmune

Abstract Background As a novel biomarker of inflammation, procalcitonin (PCT) has proven useful to guide antibiotic therapy in intensive care unit (ICU). However, there are controversial on mortality. The aim of this study was to evaluate the utility of PCT-guided antibiotic therapy in critically ill adults and determine whether studies are sufficient. Methods A systematic search in PubMed, Embase and Cochrane was performed. We included only randomized controlled trials which compared the safety and efficacy between PCT-guided or standard antibiotic therapy groups in ICU adults. Trial sequential analysis and GARDE approach were performed. Results Fifteen studies met our criteria for inclusion finally, with a cumulative number of 5486 ICU patients. There was no difference in 28-day mortality between two compared groups (P = 0.626), but significant decreases were observed in the duration of antibiotic therapy for the first episode of infection (P

Abstract During China’s urbanization process, rural labor migrants have been suggested to be one important bridge population to change urban-rural distribution on tuberculosis (TB) burden. Aiming to estimate the prevalence of TB infection and to track the active disease development in rural labor migrants, a prospective study was conducted in Shenzhen city, southern China. TB infection was detected using interferon-γ release assay (IGRA). Here we mainly report the characteristics of TB infection in the study population based on the baseline survey. A total of 4,422 eligible participants completed baseline survey in July 2013. QuantiFERON (QFT) positivity rates 17.87% (790/4,422) and was found to be consistent with the local TB epidemic of the areas where the participants immigrated from. Age, smoking, residence registered place, and present of BCG scars were found to be independently associated with QFT positivity. Additionally, evidence for interaction between smoking and age was observed (p for likelihood ratio test

After publication of the original article [1], the authors have reported that the software developers had used a new amended version of the software called “v0.9.5.10” instead of “v0.9.5.5”, which is referred to in the original article.

ABSTRACT Recruitment of monocytes to the infection site is critical for host resistance against Mycobacterium tuberculosis. CD157 has a crucial role in neutrophil and monocyte transendothelial migration and adhesion, but its role in tuberculosis (TB) is unclear. Here, we show that both mRNA and protein levels of Cd157 are significantly increased during M. tuberculosis infection. Deficiency of Cd157 impaired host response to M. tuberculosis infection by increasing bacterial burden and inflammation in the lung in the murine TB model. In vitro experiments show that the bactericidal ability was compromised in Cd157 knockout (KO) macrophages, which was due to impaired M. tuberculosis-induced reactive oxygen species (ROS) production. We further reveal that CD157 interacts with TLR2 and PKCzeta and facilitates M. tuberculosis-induced ROS production in Cd157 KO macrophages, which resulted in enhanced M. tuberculosis killing. For the clinic aspect, we observe that the expression of CD157 decreases after effective anti-TB chemotherapy. CD157 is specifically increased in pleural fluid in tuberculous pleurisy patients compared to pneumonia and lung cancer patients. Interestingly, the levels of soluble CD157 (sCD157) correlate with human peripheral monocyte-derived macrophage bactericidal activity. Exogenous application of sCD157 could compensate for macrophage bactericidal ability and restore ROS production. In conclusion, we have identified a novel protective immune function of CD157 during M. tuberculosis infection via TLR2-dependent ROS production. Application of sCD157 might be an effective strategy for host-directed therapy against TB in those with insufficient CD157 production. IMPORTANCE Tuberculosis, a chronic bacterial disease caused by Mycobacterium tuberculosis, remains a major global health problem. CD157, a dual-function receptor and β-NAD+-metabolizing ectoenzyme, promotes cell polarization, regulates chemotaxis induced through the high-affinity fMLP receptor, and controls transendothelial migration. The role of CD157 in TB pathogenesis remains unknown. In this study, we find that both mRNA and protein levels of CD157 are significantly increased in TB. Deficiency of CD157 impaired host defense against M. tuberculosis infection both in vivo and in vitro, which is mediated by an interaction among CD157, TLR2, and PKCzeta. This interaction facilitates M. tuberculosis-induced macrophagic ROS production, which enhances macrophage bactericidal activity. Interestingly, the sCD157 level in plasma is reversibly associated with MDM M. tuberculosis killing activity. By uncovering the role of CD157 in pathogenesis of TB for the first time, our work demonstrated that application of soluble CD157 might be an effective strategy for host-directed therapy against TB.

ABSTRACT Mice deficient for granulocyte-macrophage colony-stimulating factor (GM-CSF−/−) are highly susceptible to infection with Mycobacterium tuberculosis, and clinical data have shown that anti-GM-CSF neutralizing antibodies can lead to increased susceptibility to tuberculosis in otherwise healthy people. GM-CSF activates human and murine macrophages to inhibit intracellular M. tuberculosis growth. We have previously shown that GM-CSF produced by iNKT cells inhibits growth of M. tuberculosis. However, the more general role of T cell-derived GM-CSF during infection has not been defined and how GM-CSF activates macrophages to inhibit bacterial growth is unknown. Here we demonstrate that, in addition to nonconventional T cells, conventional T cells also produce GM-CSF during M. tuberculosis infection. Early during infection, nonconventional iNKT cells and γδ T cells are the main source of GM-CSF, a role subsequently assumed by conventional CD4+ T cells as the infection progresses. M. tuberculosis-specific T cells producing GM-CSF are also detected in the peripheral blood of infected people. Under conditions where nonhematopoietic production of GM-CSF is deficient, T cell production of GM-CSF is protective and required for control of M. tuberculosis infection. However, GM-CSF is not required for T cell-mediated protection in settings where GM-CSF is produced by other cell types. Finally, using an in vitro macrophage infection model, we demonstrate that GM-CSF inhibition of M. tuberculosis growth requires the expression of peroxisome proliferator-activated receptor gamma (PPARγ). Thus, we identified GM-CSF production as a novel T cell effector function. These findings suggest that a strategy augmenting T cell production of GM-CSF could enhance host resistance against M. tuberculosis. IMPORTANCE Mycobacterium tuberculosis is the bacterium that causes tuberculosis, the leading cause of death by any infection worldwide. T cells are critical components of the immune response to Mycobacterium tuberculosis. While gamma interferon (IFN-γ) is a key effector function of T cells during infection, a failed phase IIb clinical trial and other studies have revealed that IFN-γ production alone is not sufficient to control M. tuberculosis. In this study, we demonstrate that CD4+, CD8+, and nonconventional T cells produce GM-CSF during Mycobacterium tuberculosis infection in mice and in the peripheral blood of infected humans. Under conditions where other sources of GM-CSF are absent, T cell production of GM-CSF is protective and is required for control of infection. GM-CSF activation of macrophages to limit bacterial growth requires host expression of the transcription factor PPARγ. The identification of GM-CSF production as a T cell effector function may inform future host-directed therapy or vaccine designs.

BackgroundComplement functions as an important host defense system and complement C5 and C7 have been implicated in immunopathology of tuberculosis. However, little is known about the role of other complement components in tuberculosis.MethodsComplement gene expression in peripheral blood mononuclear cells of tuberculosis patients and controls were determined using whole genome transcriptional microarray assays. The mRNA and protein levels of three C1q components, C1qA, C1qB, and C1qC, were further validated by qRT-PCR and enzyme-linked immunosorbent assay, respectively. The percentages of C1q expression in CD14 positive cells were determined by flow cytometry. Finally, C1qC protein level was quantified in the pleural fluid of tuberculosis and non-tuberculosis pleurisy.ResultsC1q expression increases significantly in the peripheral blood of patients with active tuberculosis compared to healthy controls and individuals with latent TB infection. The percentage of C1q-expressing CD14 positive cells is significantly increased in active TB patients. C1q expression in the peripheral blood correlates with sputum smear positivity in tuberculosis patients and is reduced after anti-tuberculosis chemotherapy. Notably, receiver operating characteristic analysis showed that C1qC mRNA levels in peripheral blood efficiently discriminate active from latent tuberculosis infection and healthy controls. Additionally, C1qC protein level in pleural effusion shows improved power in discriminating tuberculosis from non-tuberculosis pleurisy when compared to other inflammatory markers, such as IL-6 and TNF-α.ConclusionsC1q expression correlates with active disease in human tuberculosis. C1q could be a potential diagnostic marker to discriminate active tuberculosis from latent tuberculosis infection as well as tuberculosis pleurisy from non-tuberculosis pleurisy.

Memory T cells are composed of effector, central, and memory stem cells. Previous studies have implicated that both T-bet and Eomes are involved in the generation of effector and central memory CD8 T cells. The exact role of these transcription factors in shaping the memory T cell pool is not well understood, particularly with memory stem T cells. Here, we demonstrate that both T-bet or Eomes are required for elimination of established tumors by adoptively transferred CD8 T cells. We also examined the role of T-bet and Eomes in the generation of tumor-specific memory T cell subsets upon adoptive transfer. We showed that combined T-bet and Eomes deficiency resulted in a severe reduction in the number of effector/central memory T cells but an increase in the percentage of CD62L(high)CD44(low) Sca-1(+) T cells which were similar to the phenotype of memory stem T cells. Despite preserving large numbers of phenotypic memory stem T cells, the lack of both of T-bet and Eomes resulted in a profound defect in antitumor memory responses, suggesting T-bet and Eomes are crucial for the antitumor function of these memory T cells. Our study establishes that T-bet and Eomes cooperate to promote the phenotype of effector/central memory CD8 T cell versus that of memory stem like T cells.

T cell immunoglobulin-3 (TIM-3) has been established as a negative regulatory molecule and plays a critical role in immune tolerance. TIM-3 is upregulated in exhausted CD8(+) T cells in both chronic infection and tumor. However, the nature of TIM-3(+)CD4(+) T cells in the tumor microenvironment is unclear. This study is to characterize TIM-3 expressing lymphocytes within human lung cancer tissues and establish clinical significance of TIM-3 expression in lung cancer progression.A total of 51 human lung cancer tissue specimens were obtained from pathologically confirmed and newly diagnosed non-small cell lung cancer (NSCLC) patients. Leukocytes from tumor tissues, distal normal lung tissues, and peripheral blood mononuclear cells (PBMC) were analyzed for TIM-3 surface expression by flow cytometry. TIM-3 expression on tumor-infiltrating lymphocytes (TILs) was correlated with clinicopathological parameters.TIM-3 is highly upregulated on both CD4(+) and CD8(+) TILs from human lung cancer tissues but negligibly expressed on T cells from patients' peripheral blood. Frequencies of IFN-γ(+) cells were reduced in TIM-3(+)CD8(+) TILs compared to TIM-3(-)CD8(+) TILs. However, the level of TIM-3 expression on CD8(+) TILs failed to associate with any clinical pathological parameter. Interestingly, we found that approximately 70% of TIM-3(+)CD4(+) TILs expressed FOXP3 and about 60% of FOXP3(+) TILs were TIM-3(+). Importantly, TIM-3 expression on CD4(+) T cells correlated with poor clinicopathological parameters of NSCLC such as nodal metastasis and advanced cancer stages. Our study reveals a new role of TIM-3 as an important immune regulator in the tumor microenvironment via its predominant expression in regulatory T cells.

BACKGROUND: Despite great efforts to improve diagnosis and treatment, tuberculosis (TB) remains a major health problem worldwide, especially in developing countries. Lack of concrete immune markers is still the obstacle to properly evaluate active TB. Therefore, identification of more validated biomarkers and phenotypic signatures is imperative. In particular, T cell-related biomarkers are more significant. METHODOLOGY: To understand the nature of CD4(+) T cell-derived signatures involved in infection and disease development, we examined and analyzed whole genome expression profiles of purified CD4(+) T cells from healthy individuals (HD), two distinct populations with latent infection (with low or high IFN-γ levels, LTB(L)/LTB(H)) and untreated TB patients. Following, we validated the expression profiles of genes in the peripheral CD4(+) T cells from each group and examined secretion levels of distinct cytokines in serum and pleural effusion. PRINCIPAL FINDINGS: Our bio-informatic analyses indicate that the two latent populations and clinical TB patients possess distinct CD4(+) T cell gene expression profiles. Furthermore, The mRNA and protein expression levels of B cell activating factor (BAFF), which belongs to the TNF family, and a proliferation-inducing ligand (APRIL) were markedly up-regulated at the disease stage. In particular, the dramatic enhancement of BAFF and APRIL in the pleural effusion of patients with tuberculosis pleurisy suggests that these proteins may present disease status. In addition, we found that the BAFF/APRIL system was closely related to the Th1 immune response. Our study delineates previously unreported roles of BAFF and APRIL in the development of tuberculosis, and these findings have implications for the diagnosis of the disease. Our study also identifies a number of transcriptional signatures in CD4(+) T cells that have the potential to be utilized as diagnostic and prognostic tools to combat the tuberculosis epidemic.