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Exhausted CD8 T cells (TEX) are a distinct state of T cell differentiation associated with failure to clear chronic viruses and cancer. Immunotherapies such as PD-1 blockade can reinvigorate TEX cells, but reinvigoration is not durable. A major unanswered question is whether TEX cells differentiate into functional durable memory T cells (TMEM) upon antigen clearance. Here, using a mouse model, we found that upon eliminating chronic antigenic stimulation, TEX cells partially (re)acquire phenotypic and transcriptional features of TMEM cells. These ‘recovering’ TEX cells originated from the T cell factor (TCF-1+) TEX progenitor subset. Nevertheless, the recall capacity of these recovering TEX cells remained compromised as compared to TMEM cells. Chromatin-accessibility profiling revealed a failure to recover core memory epigenetic circuits and maintenance of a largely exhausted open chromatin landscape. Thus, despite some phenotypic and transcriptional recovery upon antigen clearance, exhaustion leaves durable epigenetic scars constraining future immune responses. These results support epigenetic remodeling interventions for TEX cell–targeted immunotherapies. Wherry and colleagues examine whether exhausted T cells (TEX) can differentiate into functional memory T cells (TMEM) when chronic antigen is withdrawn. Using the chronic LCMV infection mouse model, they show that ‘recovering’ TEX cells (REC-TEX) only partially recover immunophenotypic and functional characteristics of TMEM cells. The epigenomic status of REC-TEX cells more closely resembles that of TEX cells, and, upon rechallenge, the REC-TEX cells were still compromised in their ability to respond to virus.

Mycobacterium bovis bacille Calmette-Guérin (BCG) has been used for 100 years and prevents disseminated tuberculosis and death in young children. However, it shows only partial efficacy against pulmonary tuberculosis (TB) in adults, so new vaccines are urgently needed. The protective efficacy of BCG depends on T cells, which are typically activated by pathogen-derived protein antigens that bind to highly polymorphic major histocompatibility complex (MHC) molecules. Some T cells recognize non-protein antigens via antigen presenting systems that are independent of genetic background, leading to their designation as donor-unrestricted T (DURT) cells. Whether live whole cell vaccines, like BCG, can induce durable expansions of DURT cells in humans is not known. We used combinatorial tetramer staining, multi-parameter flow cytometry, and immunosequencing to comprehensively characterize the effect of BCG on activation and expansion of DURT cell subsets. We examined peripheral blood mononuclear cells (PBMC) derived from a Phase I study of South African adults in which samples were archived at baseline, 3 weeks, and 52 weeks post-BCG revaccination. We did not observe a change in the frequency of total mucosal-associated invariant T (MAIT) cells, invariant natural killer T (iNKT) cells, germline encoded mycolyl-reactive (GEM) T cells, or γδ T cells at 52 weeks post-BCG. However, immunosequencing revealed a set of TCR-δ clonotypes that were expanded at 52 weeks post-BCG revaccination. These expanded clones expressed the Vδ2 gene segment and could be further defined on the basis of biochemical similarity into several ‘meta-clonotypes’ that likely recognize similar epitopes. Our data reveal that BCG vaccination leads to durable expansion of DURT cell clonotypes despite a limited effect on total circulating frequencies in the blood and have implications for defining the immunogenicity of candidate whole cell TB vaccines.

Key Points MAIT cells comprise half the CD8 T cell IFN-γ response to BCG in blood. Frequencies of MAIT cells were not sustainably modulated by BCG vaccination. Innate cytokines mediate BCG-induced MAIT cell responses in whole blood., Tuberculosis (TB) is the leading cause of mortality from a single infectious agent, Mycobacterium tuberculosis. Relevant immune targets of the partially efficacious TB vaccine bacille Calmette–Guérin (BCG) remain poorly defined. Mucosal-associated invariant T (MAIT) cells are MHC-related protein 1 (MR1)–restricted T cells, which are reactive against M. tuberculosis, and underexplored as potential TB vaccine targets. We sought to determine whether BCG vaccination activated mycobacteria-specific MAIT cell responses in humans. We analyzed whole blood samples from M. tuberculosis–infected South African adults who were revaccinated with BCG after a six-month course of isoniazid preventative therapy. In vitro BCG stimulation potently induced IFN-γ expression by phenotypic (CD8+CD26+CD161+) MAIT cells, which constituted the majority (75%) of BCG-reactive IFN-γ–producing CD8+ T cells. BCG revaccination transiently expanded peripheral blood frequencies of BCG-reactive IFN-γ+ MAIT cells, which returned to baseline frequencies a year following vaccination. In another cohort of healthy adults who received BCG at birth, 53% of mycobacteria-reactive–activated CD8 T cells expressed CDR3α TCRs, previously reported as MAIT TCRs, expressing the canonical TRAV1-2-TRAJ33 MAIT TCRα rearrangement. CD26 and CD161 coexpression correlated with TRAV1-2+CD161+ phenotype more accurately in CD8+ than CD4−CD8− MAIT cells. Interestingly, BCG-induced IFN-γ expression by MAIT cells in vitro was mediated by the innate cytokines IL-12 and IL-18 more than MR1-induced TCR signaling, suggesting TCR-independent activation. Collectively, the data suggest that activation of blood MAIT cells by innate inflammatory cytokines is a major mechanism of responsiveness to vaccination with whole cell vaccines against TB or in vitro stimulation with mycobacteria (Clinical trial registration: NCT01119521).

Whether conventional dendritic cells (cDC) acquire subset identity under direction of Wnt family glycoproteins is unknown. We demonstrate that Wnt4, a β-catenin–independent Wnt ligand, is produced by both hematopoietic and nonhematopoietic cells and is both necessary and sufficient for preconventional DC1/cDC1 maintenance. Whereas bone marrow cDC precursors undergo phosphoJNK/c-Jun activation upon Wnt4 treatment, loss of cDC Wnt4 in CD11cCreWnt4flox/flox mice impaired differentiation of CD24+, Clec9A+, CD103+ cDC1 compared with CD11cCre controls. Conversely, single-cell RNA sequencing analysis of bone marrow revealed a 2-fold increase in cDC2 gene signature genes, and flow cytometry demonstrated increased numbers of SIRP-α+ cDC2 amid lack of Wnt4. Increased cDC2 numbers due to CD11c-restricted Wnt4 deficiency increased IL-5 production, group 2 innate lymphoid cell expansion, and host resistance to the hookworm parasite Nippostrongylus brasiliensis. Collectively, these data uncover a novel and unexpected role for Wnt4 in cDC subset differentiation and type 2 immunity.

SummaryAntigen classes other than proteins can be presented to T cells by near-monomorphic antigen-presenting molecules such as CD1, MR1, and butyrophilin 3A1. We sought to define the roles of donor unrestricted T (DURT) cells, including MR1-reactive MAIT cells, CD1b-reactive glucose monomycolate (GMM)-specific T cells, CD1d-reactive NKT cells, and γδ T cells, in vaccination against Mycobacterium tuberculosis. We characterized DURT cells following primary bacille Calmette-Guerin (BCG) vaccination in infants or BCG-revaccination in adults. BCG (re)vaccination did not modulate peripheral blood frequencies, T cell activation or memory profiles of MAIT cells, CD1b-restricted GMM-specific and germline-encoded mycolyl-reactive (GEM) cells or CD1d- restricted NKT cells. By contrast, BCG vaccination was associated with increased frequencies of γδ T cells as well as a novel subset of IFN-γ-expressing CD4+ T cells with a CD26+CD161+TRAV1-2− phenotype in infants. More studies are required to understand the full potential of DURT cells in new TB vaccine strategies.

Adjuvants are critical for improving the quality and magnitude of adaptive immune responses to vaccination. Lipid nanoparticle (LNP)-encapsulated nucleoside-modified mRNA vaccines have shown great efficacy against SARS-CoV-2, but the mechanism of action of this vaccine platform is not well-characterized. Using influenza virus and SARS-CoV-2 mRNA and protein subunit vaccines, we demonstrated that our LNP formulation has intrinsic adjuvant activity that promotes the induction of strong T follicular helper cell, germinal center B cell, long-lived plasma cell and memory B cell responses that are associated with durable and protective antibodies in mice. Comparative experiments demonstrated that this LNP outperformed a widely used MF59-like adjuvant, AddaVax™. The adjuvant activity of the LNP relies on the ionizable lipid component and on IL-6 cytokine induction, but not on MyD88- or MAVS-dependent sensing of LNPs. Our study identified LNPs as a versatile adjuvant that enhances the efficacy of both traditional and next-generation vaccine platforms., Graphical Abstract, The mechanism of action of nucleoside-modified mRNA-LNP vaccines is unknown. Alameh, Tombácz, Bettini et al. demonstrate that LNPs can possess adjuvant activity and promote a robust induction of Tfh cell, B cell and humoral responses when utilized in mRNA and protein subunit vaccines in mice. IL-6 induction and the ionizable lipid component are critical for the adjuvant activity of LNPs.

TCF-1 is a key transcription factor in progenitor exhausted CD8 T cells (Tex). Moreover, this Tex cell subset mediates responses to PD-1 checkpoint pathway blockade. However, the role of the transcription factor TCF-1 in early fate decisions and initial generation of Tex cells is unclear. Single cell RNA-sequencing (scRNA-seq) and lineage tracing identified a TCF-1(+)Ly108(+)PD-1(+) CD8 T cell population early during chronic infection that seeds development of mature Tex cells. TCF-1 mediated the bifurcation between divergent fates, repressing development of terminal KLRG1(Hi) effectors while fostering KLRG1(Lo) Tex precursor cells, and PD-1 stabilized this TCF-1(+) Tex precursor cell pool. TCF-1 mediated a T-bet to Eomes transcription factor transition in Tex precursors by promoting Eomes expression and drove c-Myb expression that controlled Bcl-2 and survival. These data define a role for TCF-1 in early fate bifurcation driving Tex precursor cells, and also identify PD-1 as a protector of this early TCF-1 subset.

Biomarkers for TB treatment response and outcome are needed. This study characterize changes in immune profiles during TB treatment, define biosignatures associated with treatment outcomes, and explore the feasibility of predictive models for relapse. Seventy-two markers were measured by multiplex cytokine array in serum samples from 78 cured, 12 relapsed and 15 failed treatment patients from South Africa before and during therapy for pulmonary TB. Promising biosignatures were evaluated in a second cohort from Uganda/Brazil consisting of 17 relapse and 23 cured patients. Thirty markers changed significantly with different response patterns during TB treatment in cured patients. The serum biosignature distinguished cured from relapse patients and a combination of two clinical (time to positivity in liquid culture and BMI) and four immunological parameters (TNF-β, sIL-6R, IL-12p40 and IP-10) at diagnosis predicted relapse with a 75% sensitivity (95%CI 0.38-1) and 85% specificity (95%CI 0.75-0.93). This biosignature was validated in an independent Uganda/Brazil cohort correctly classifying relapse patients with 83% (95%CI 0.58-1) sensitivity and 61% (95%CI 0.39-0.83) specificity. A characteristic biosignature with value as predictor of TB relapse was identified. The repeatability and robustness of these biomarkers require further validation in well-characterized cohorts.

Tuberculosis kills more people than any other infectious disease. Three pivotal trials testing 4-month regimens failed to meet non-inferiority margins; however, approximately four-fifths of participants were cured. Through a pooled analysis of patient-level data with external validation, we identify populations eligible for 4-month treatment, define phenotypes that are hard to treat and evaluate the impact of adherence and dosing strategy on outcomes. In 3,405 participants included in analyses, baseline smear grade of 3+ relative to2+, HIV seropositivity and adherence of ≤90% were significant risk factors for unfavorable outcome. Four-month regimens were non-inferior in participants with minimal disease defined by2+ sputum smear grade or non-cavitary disease. A hard-to-treat phenotype, defined by high smear grades and cavitation, may require durations6 months to cure all. Regimen duration can be selected in order to improve outcomes, providing a stratified medicine approach as an alternative to the 'one-size-fits-all' treatment currently used worldwide.

One third of the global population is estimated to be latently infected with Mycobacterium tuberculosis. We performed a phase I randomized controlled trial of isoniazid preventive therapy (IPT) before revaccination with bacillus Calmette–Guérin (BCG) in healthy, tuberculin skin test–positive (≥15-mm induration), HIV-negative South African adults. We hypothesized that preclearance of latent bacilli with IPT modulates BCG immunogenicity following revaccination. Frequencies and coexpression of IFN-γ, TNF-α, IL-2, IL-17, and/or IL-22 in CD4 T cells and IFN-γ–expressing CD8 T, γδ T, CD3+CD56+ NKT-like, and NK cells in response to BCG were measured using whole blood intracellular cytokine staining and flow cytometry. We analyzed 72 participants who were revaccinated with BCG after IPT (n = 33) or without prior IPT (n = 39). IPT had little effect on frequencies or cytokine coexpression patterns of M. tuberculosis– or BCG-specific responses. Revaccination transiently boosted BCG-specific Th1 cytokine-expressing CD4, CD8, and γδ T cells. Despite high frequencies of IFN-γ–expressing BCG-reactive CD3+CD56+ NKT-like cells and CD3−CD56dim and CD3−CD56hi NK cells at baseline, BCG revaccination boosted these responses, which remained elevated up to 1 y after revaccination. Such BCG-reactive memory NK cells were induced by BCG vaccination in infants, whereas in vitro IFN-γ expression by NK cells upon BCG stimulation was dependent on IL-12 and IL-18. Our data suggest that isoniazid preclearance of M. tuberculosis bacilli has little effect on the magnitude, persistence, or functional attributes of lymphocyte responses boosted by BCG revaccination. Our study highlights the surprising durability of BCG-boosted memory NKT-like and NK cells expressing antimycobacterial effector molecules, which may be novel targets for tuberculosis vaccines.

B cell subsets expressing the transcription factor T-bet are associated with humoral immune responses and autoimmunity. Here we examined the anatomic distribution, clonal relationships, and functional properties of T-bet(+) and T-bet(−) memory B cells (MBCs) in the context of the influenza-specific immune response. In mice, both T-bet(−) and T-bet(+) hemagglutinin-specific B cells arose in germinal centers, acquired memory B cell markers, and persisted indefinitely. Lineage tracing and IgH repertoire analyses revealed minimal interconversion between T-bet(−) and T-bet(+) MBCs, and parabionts showed differential tissue residency and recirculation properties. T-bet(+) MBCs could be subdivided into recirculating T-bet(lo) MBCs and spleen-resident T-bet(hi) MBCs. Human MBCs displayed similar features. Conditional gene deletion studies revealed that T-bet expression in B cells was required for nearly all HA stalk-specific IgG2c antibodies and for durable neutralizing titers to influenza. Thus, T-bet expression distinguishes MBC subsets that have profoundly different homing, residency, and functional properties, and mediate distinct aspects of humoral immune memory.

A long-standing question in immunology has been to understand how transcription factors (TF) determine cell-type-specific transcription programs. Traditionally, investigating TFs in immune cells has been limited to measuring the effect of a single TF on a limited number of gene targets. The advent of next generation sequencing methods makes it possible to measure the effects of multiple transcription factors on a genome-wide scale. This holistic approach gives us a better understanding of the influence that multiple TFs have on cell-specific programs. In this issue of European Journal of Immunology, Fang et al. [Eur. J. Immunol. 2015. 45: 3150-3157] show that genomic regions cooccupied with two or more TFs show increased epigenetic histone marks of active enhancers, which correspond to increased transcriptional activity.

Great progress has been made in understanding immunity to viral infection. However, infection can occur in the context of co-infection by unrelated pathogens that modulate immune responses and/or disease. We have studied immunity and disease during co-infection with two unrelated viruses: Ectromelia virus (ECTV) and Lymphocytic Choriomeningitis virus (LCMV). ECTV infection can be a lethal in mice due in part to the blockade of Type I Interferons (IFN-I). We show that ECTV/LCMV co-infection results in decreased ECTV viral load and amelioration of ECTV-induced disease, likely due to IFN-I induction by LCMV, as rescue is not observed in IFN-I receptor deficient mice. However, immune responses to LCMV in ECTV co-infected mice were also lower compared to mice infected with LCMV alone and potentially biased toward effector-memory cell generation. Thus, we provide evidence for bi-directional effects of viral co-infection that modulate disease and immunity.

Phase 2 clinical trials for tuberculosis (TB) treatment require reliable culture methods to determine presence or absence of Mycobacterium tuberculosis (Mtb) over the course of therapy, as these trials are based primarily on bacteriological endpoints. We evaluate which of 5 solid media is most reliable: Lowenstein-Jensen (LJ) egg-based medium and 4 Middlebrook agar media (nonselective 7H10 and 7H11 and selective 7H10 and 7H11). We analyze 393 specimens from 50 HIV-negative Ugandan adults with newly-diagnosed, pulmonary TB and high acid-fast bacillus smear grade. Specimens were collected every 2–4 weeks during the first 12 weeks of therapy. We compare the results for each culture to 2 composite reference standards—one that was deemed positive if any solid culture was positive for Mtb and another based on latent class analysis. Both reference standards established that the 2 selective Middlebrook media most reliably determine the presence or absence of Mtb (P

Background T-cell interferon-γ release assays (IGRAs) are used in the diagnosis of Mycobacterium tuberculosis infection and could be useful biomarkers of response to treatment of latent TB infection for clinical trials, infection control units, and TB programs. Methods This investigation was a prospective, controlled substudy of IGRA responses in 82 healthy South African adults with HIV seronegative and positive tuberculin skin test results randomly assigned to treatment with 6 months of daily isoniazid preventive therapy (IPT) or observation before Bacillus Calmette-Guerin revaccination in a clinical trial. QuantiFERON-TB Gold In-Tube (QFT-GIT) assay was used to measure interferon-γ (IFN-γ) response to mycobacterial antigens at baseline and after IPT or observation. Results IFN-γ levels declined between baseline and the end of IPT (signed rank test P ≤ .0001) and between baseline and a similar period of observation without IPT (signed rank test P = .03). The rate of decrease in IFN-γ responses over time did not differ between the groups (Mann-Whitney-Wilcoxon test P = .31). QFT-GIT test results in two subjects (5%) in the IPT group and two subjects (5%) in the observation group reverted from positive to negative during follow-up. No significant difference was found between the groups with respect to baseline positivity or the proportion of patients whose tests reverted to negative. Conclusions IPT had no effect on changes in QFT-GIT readouts during short-term follow-up of adults with positive tuberculin skin tests in a high TB incidence setting. QFT-GIT is unlikely to be a useful biomarker of response to treatment of latent TB infection. Trial registry ClinicalTrials.gov ; No.: NCT01119521; URL: www.clinicaltrials.gov

Whether conventional dendritic cells (cDC) acquire subset identity under direction of regenerative glycoproteins is unknown. We demonstrate that Wnt4, a beta-catenin independent Wnt ligand, is necessary and sufficient for pre-cDC1 BM specification. CD11c-restricted Wnt4 deficiency reduced mature cDC1 numbers in BM, spleen, lung, and intestine and Wnt4 directly induced of pJNK activation and cDC1 commitment. CD11cCreWnt4flox/flox mice lacked IRF8/cJun complex stabilization and had increased basal numbers of cDC2, group 2 innate lymphoid cells ILC2 and increased resistance to the parasite Nippostrongylus brasiliensis compared to CD11cCre controls. These data reveal a novel role for Wnt4 in DC commitment and pathogen-specific immunity.

T-cell exhaustion is a hallmark of immunologic failure in chronic infections and cancer. Blocking immune inhibitory receptors such as programmed death-1 (PD-1) can re-invigorate exhausted T-cells (Tex), but many patients fail to achieve durable disease control. Thus, a deeper understanding of molecular pathways underlying reversal of T-cell exhaustion is needed. Little is known about “reprogramming” of Tex into recovered T-cells (Trecov) with better functionality and durable memory properties following cure of chronic disease. Here, we aim to determine the molecular program and underlying mechanisms of Trecov. We used the well-defined LCMV mouse model of T-cell exhaustion. To achieve “cure” of chronic infection, we adoptively transferred Tex from mice chronically infected with LCMV clone 13 into antigen-free mice and interrogated changes in Tex as they recovered, including testing the recall capacity of Trecov, a quintessential memory T-cell (Tmem) property. These studies revealed some recovery of phenotypic markers of T-cell memory, but also demonstrated that “scars” of chronic infection persisted. For example, although expression of the IL-7R was increased and PD-1 expression was lower, consistent with differentiation toward memory, the frequency of IL-7R+ cells was lower and PD-1 expression was still higher when compared to bona fide memory T-cells. These changes were accompanied by partial recovery from dysfunction, where Trecov displayed a moderate improvement in effector function. Consistent with this partial recovery, single-cell RNA sequencing (scRNAseq) indicated that the gene-expression profile of Trecov resembles Tmem in some respects, but other features were still more similar to Tex cells. To test how this collection of changes impacted a key property of Tmem, the ability to mount a recall response, we designed a series of challenge experiments. These studies revealed that although Trecov responded better than Tex, recall capacity was still inferior on a per cell basis compared to true Tmem. Together, these results suggest that following elimination of persistent antigenic stimulation and inflammation, previously exhausted T-cells are able to recover some properties of Tmem, while other aspects remain “scarred” from their history of exhaustion. These studies will enhance our understanding of immunologic and molecular mechanisms of Tex recovery, and have the potential to identify novel therapeutic strategies for recovery of more durable functional T-cells in the treatment of cancer and chronic infection. Citation Format: Mohamed S. Abdel-Hakeem, Jean-Christophe Beltra, Zeyu Chen, John Johnson, Saskinath Manne, Mohammed-Alkhatim Ali, E. John Wherry. Reprogramming of exhausted T-cells following cure of chronic viral infection [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr A166.

We retrospectively analyzed time to detection of 3747 positive MGIT sputum cultures at a laboratory in a country with heavy burden of tuberculosis. Ninety-nine percent of diagnostic cultures turned positive within 28 days, suggesting that physicians may consider alternative diagnoses if sputum cultures remain negative after 4 weeks of incubation.

Standard methods to monitor tuberculosis (TB) treatment response rely on sputum microscopy and culture conversion. Alternatives to these methods are needed for those patients whose sputum tests are smear or culture negative. Here, we examine anti-phospholipid IgM antibody level changes as a biomarker for treatment response in smear positive TB patients. Serum samples were obtained from 40 pulmonary TB patients at the start and end of the intensive phase treatment (IPT) from the CDC-TB Trials Consortium randomized clinical trial in Kampala, Uganda. Samples were screened by ELISA for IgM levels against five phospholipids found in Mycobacterium tuberculosis and host cells. Lipid antigens included cardiolipin (CL), phosphatidyl inositol (PI), phosphatidyl ethanolamine (PE), phosphatidyl choline (PTC), and sphingolipid (SL). Levels of IgM against all phospholipids significantly decreased (p = 0.034, 0.001, 0.008 0.008, 0.040, respectively) following anti-TB drug treatment in patients without lung cavitary disease at baseline. The mean sensitivity of this test in these patients was 83% when the IgM response to a single lipid antigen was used; it was >90% when responses to 2 or more lipids were assessed. In contrast, cavitary TB patients showed an overall IgM increase, with a significant rise against PE (p = 0.025). There was no significant difference in the change in antibody levels between patients who remained culture-positive and those who culture-converted after 40 doses of drug therapy. The measurement of IgM anti-phospholipid antibodies may be a useful biomarker to monitor treatment response in non-cavitary TB patients.

International audience; T cell development is orchestrated by transcription factors that regulate the expression of genes initially buried within inaccessible chromatin, but the transcription factors that establish the regulatory landscape of the T cell lineage remain unknown. Profiling chromatin accessibility at eight stages of T cell development revealed the selective enrichment of TCF-1 at genomic regions that became accessible at the earliest stages of development. TCF-1 was further required for the accessibility of these regulatory elements and at the single-cell level, it dictated a coordinate opening of chromatin in T cells. TCF-1 expression in fibroblasts generated de novo chromatin accessibility even at chromatin regions with repressive marks, inducing the expression of T cell-restricted genes. These results indicate that a mechanism by which TCF-1 controls T cell fate is through its widespread ability to target silent chromatin and establish the epigenetic identity of T cells.

Linezolid, the first oxazolidinone approved for clinical use, has effective in vitro and promising in vivo activity against Mycobacterium tuberculosis.To evaluate the early and extended early bactericidal activity of linezolid in patients with pulmonary tuberculosis.Randomized open label trial. Thirty patients with newly diagnosed smear-positive pulmonary tuberculosis (10 per arm) were assigned to receive isoniazid (300 mg daily) and linezolid (600 mg twice daily or 600 mg once daily) for 7 days. Sputum for quantitative culture was collected for 2 days before and then daily during 7 days of study drug administration. Bactericidal activity was estimated by measuring the decline in bacilli during the first 2 days (early bactericidal activity) and the last 5 days of study drug administration (extended early bactericidal activity).The mean early bactericidal activity of isoniazid (0.67 log10 cfu/ml/d) was greater than that of linezolid twice and once daily (0.26 and 0.18 log10 cfu/ml/d, respectively). The extended early bactericidal activity of linezolid between Days 2 and 7 was minimal.Linezolid has modest early bactericidal activity against rapidly dividing tubercle bacilli in patients with cavitary pulmonary tuberculosis during the first 2 days of administration, but little extended early bactericidal activity. Clinical trial registered with www.clinicaltrials.gov (NCT00396084).

Biomarkers for monitoring response to anti-tuberculosis treatment are needed. We explored immune markers previously published as having predictive capability for 8 week culture status in 39 adults enrolled in a clinical trial in Kampala, Uganda.We consecutively selected 20 HIV-negative pulmonary TB subjects with positive cultures, and 19 subjects with negative cultures at the end of intensive phase therapy. At baseline and after 8 weeks, serum was assayed for nine cytokines and soluble cytokine receptors using multiplexed platforms or ELISA. We evaluated their association with week 8 culture status first using single-variable logistic models, then using cross-validated estimates of the C-statistic, a measure of discrimination, of candidate models including 2 or 3 analytes in addition to age.All but one analyte decreased from baseline to week 8 (all p 0.01). Individual biomarkers were not associated with 8 week culture status. Logistic models including increasing age, higher baseline soluble tumor necrosis factor receptor alpha 1 (sTNF-R1), and higher week 8 C-reactive protein (CRP) concentration classified subjects by culture status with up to 85% accuracy and acceptable discrimination (cross-validated C-statistic 0.76) and calibration (Hosmer-Lemeshow P 0.2).Exploratory post-hoc models including sTNF-R1, CRP, and age, classified 8 week culture status with promising accuracy.

Tuberculosis (TB) is the most common life-threatening infection in human immunodeficiency virus (HIV)-infected persons and frequently occurs before the onset of severe immunodeficiency. Development of TB is associated with increased HIV type 1 (HIV-1) viral load, a fall in CD4 lymphocyte counts, and increased mortality. The aim of this study was to examine how treatment of pulmonary TB affected HIV-1 activity in HIV-1/TB-coinfected subjects with CD4 cell counts of >100 cells/μl. HIV-1/TB-coinfected subjects were recruited in Kampala, Uganda, and were monitored over time. Based upon a significant (0.5 log10copies/ml) decrease in viral load by the end of treatment, two patient groups could be distinguished. Responders (n= 17) had more rapid resolution of anemia and pulmonary lesions on chest radiography during TB treatment. This group had a significant increase in viral load to levels not different from those at baseline 6 months after completion of TB treatment. HIV-1 viral load in nonresponders (n= 10) with TB treatment increased and at the 6 month follow-up was significantly higher than that at the time of diagnosis of TB. Compared to baseline levels, serum markers of macrophage activation including soluble CD14 decreased significantly by the end of TB treatment in responders but not in nonresponders. These data further define the impact of pulmonary TB on HIV-1 disease. HIV-1 replication during dual HIV-1/TB infection is not amenable to virologic control by treatment of TB alone. Concurrent institution of highly active antiretroviral treatment needs to be evaluated in patients dually infected with pulmonary TB and HIV-1.

Biologically active transforming growth factor beta 1 (TGFbeta1) has been identified at sites of Mycobacterium tuberculosis (MTB) infection in the lung; however, the underlying mechanism(s) for its activation is not clear. Here using an enzyme-linked immunospot assay for TGFbeta1, we show that human blood monocytes (MN) and alveolar macrophages (AM) produce bioactive TGFbeta1 upon stimulation by MTB. However, only MTB-stimulated MN increased TGFbeta1 production on a per cell basis. The frequency of TGFbeta1-producing MN was reduced by an inhibitor of plasmin, bdellin, indicating a role for plasmin pathways in the bioactivation of cytokine. The expression of urokinase plasminogen activator receptor (uPAR) mRNA and both surface and soluble uPAR (CD87) was increased in MTB-activated MN. However, antibody neutralization of uPAR suppressed bioactive TGFbeta1 in MN alone. Thus, the more immature MN, which are continuously recruited to the lung during tuberculosis (TB), have a higher capacity to bioactivate TGFbeta1 by expression of components of the plasmin pathway. Excess production and bioactivation of TGFbeta1 at sites of MTB infection may undermine host immune responses during TB.

Background. Active tuberculosis may accelerate progression of human immunodeficiency virus (HIV) infection by promoting viral replication in activated lymphocytes. Glucocorticoids are used in pleural tuberculosis to reduce inflammation-induced pathology, and their use also might reduce progression of HIV by suppressing immune activation. We examined the effect that prednisolone has on survival in HIV-1-associated pleural tuberculosis. Methods. We conducted a randomized, double-blind, placebo-controlled trial of prednisolone as an adjunct to tuberculosis treatment, in adults with HIV-1-associated pleural tuberculosis. The primary outcome was death. Analysis was by intention to treat. Results. Of 197 participants, 99 were assigned to the prednisolone group and 98 to the placebo group. The mortality rate was 21 deaths/100 person-years (pyr) in the prednisolone group and 25 deaths/100 pyr in the placebo group (age-, sex-, and initial CD4 + T cell count-adjusted mortality rate ratio, 0.99 [95% confidence interval, 0.62-1.56] [P = .95]). Resolution of tuberculosis was faster in the prednisolone group, but recurrence rates were slightly (though not significantly) higher, and use of prednisolone was associated with a significantly higher incidence of Kaposi sarcoma (4.2 cases/100 pyr, compared with 0 cases/100 pyr [P = .02]). Conclusions. In view of the lack of survival benefit and the increased risk of Kaposi sarcoma, the use of prednisolone in HIV-associated tuberculous pleurisy is not recommended.

Background Infection of alveolar macrophages (AMs), which constitute the first line of defense against Mycobacterium tuberculosis, initiates an intense interaction between the host's innate immune response and mycobacteria that may assist in the successful intracellular parasitism of M. tuberculosis. Methods Expression of tumor necrosis factor (TNF)- alpha and M. tuberculosis 85B mRNA was studied in M. tuberculosis-infected AMs, to better delineate the role of macrophages in the early events in initiation of infection. Results Both TNF- alpha mRNA and M. tuberculosis 85B were induced in AMs; at 24 h, the time point of maximum TNF- alpha induction, the mRNA levels for TNF- alpha and M. tuberculosis 85B correlated with one another, and induction of either gene correlated strongly with their protein levels. Inhibition of endogenous TNF- alpha by soluble (s) TNF receptor (R) I and sTNFRII reduced expression of both TNF- alpha and M. tuberculosis 85B. The activation of nuclear factor- kappa B was found to underlie expression of both TNF- alpha and M. tuberculosis 85B. Exogenous TNF- alpha was slightly more potent than interleukin (IL)-6 and granulocyte-macrophage colony-stimulating factor and was significantly stronger than IL-1 in inducing expression of M. tuberculosis 85B. Interestingly, inhibition of bactericidal mediators, reactive oxygen intermediates (ROIs) and reactive nitrogen intermediates (RNIs), reduced expression of TNF- alpha and M. tuberculosis 85B genes in M. tuberculosis-infected AMs. Conclusion Activation of AMs by M. tuberculosis initiates a cascade of events whereby TNF- alpha, ROI, and RNI enhance the expression of the M. tuberculosis 85B gene.

The timely evaluation of new drugs that can be used to shorten tuberculosis (TB) treatment will require surrogate markers for relapse. This study examined bactericidal activity against intracellular Mycobacterium tuberculosis in whole blood culture (whole blood bactericidal activity; WBA) during TB treatment. In the absence of chemotherapy, immune mechanisms in patient blood resulted in bacteriostasis, whereas administration of oral chemotherapy resulted in bacillary killing. Total WBA per dose was greater during the intensive phase of treatment than during the continuation phase (mean, -2.32 vs. -1.67 log(10) cfu-days, respectively; P

Sputum and serum from patients with active pulmonary tuberculosis (TB), healthy purified protein derivative-positive adults, and patients with bacterial pneumonia were collected to simultaneously assess local immunity in the lungs and peripheral blood. To determine whether cytokine profiles in sputum from TB patients and control subjects were a reflection of its cellular composition, cytospin slides were prepared in parallel and assessed for the presence of relative proportions of epithelial cells, neutrophils, macrophages, and T cells. Gamma interferon (IFN-γ) in sputum from TB patients was markedly elevated over levels for both control groups. With anti-TB therapy, IFN-γ levels in sputum from TB patients decreased rapidly and by week 4 of treatment were comparable to those in sputum from controls. Further, IFN-γ levels in sputum closely followed mycobacterial clearance. Although detected at fourfold-lower levels, IFN-γ immunoreactivities in serum followed kinetics in sputum. TNF-α, interleukin 8 (IL-8) and IL-6 also were readily detected in sputum from TB patients at baseline and responded to anti-TB therapy. In contrast to IFN-γ, however, TNF-α and IL-8 levels also were elevated in sputum from pneumonia controls. These data indicate that sputum cytokines correlate with disease activity during active TB of the lung and may serve as potential early markers for sputum conversion and response to anti-TB therapy.

Our understanding of mechanisms underlying progression from Mycobacterium tuberculosis infection to pulmonary tuberculosis disease in humans remains limited. To define such mechanisms, we followed M. tuberculosis-infected adolescents longitudinally. Blood samples from forty-four adolescents who ultimately developed tuberculosis disease (“progressors”) were compared with those from 106 matched controls, who remained healthy during two years of follow up. We performed longitudinal whole blood transcriptomic analyses by RNA sequencing and plasma proteome analyses using multiplexed slow off-rate modified DNA aptamers. Tuberculosis progression was associated with sequential modulation of immunological processes. Type I/II interferon signalling and complement cascade were elevated 18 months before tuberculosis disease diagnosis, while changes in myeloid inflammation, lymphoid, monocyte and neutrophil gene modules occurred more proximally to tuberculosis disease. Analysis of gene expression in purified T cells also revealed early suppression of Th17 responses in progressors, relative to M. tuberculosis-infected controls. This was confirmed in an independent adult cohort who received BCG re-vaccination; transcript expression of interferon response genes in blood prior to BCG administration was associated with suppression of IL-17 expression by BCG-specific CD4 T cells 3 weeks post-vaccination. Our findings provide a timeline to the different immunological stages of disease progression which comprise sequential inflammatory dynamics and immune alterations that precede disease manifestations and diagnosis of tuberculosis disease. These findings have important implications for developing diagnostics, vaccination and host-directed therapies for tuberculosis. Trial registration Clincialtrials.gov, NCT01119521, Author summary To define biological mechanisms that underlie progression of Mycobacterium tuberculosis infection to active tuberculosis, we followed M. tuberculosis-infected adolescents longitudinally. Those who ultimately developed tuberculosis disease (“progressors”) were compared with matched controls, who remained healthy. Whole blood transcriptomic and plasma proteome analyses showed sequential modulation of immunological processes. Type I/II interferon signalling and complement cascade were elevated 18 months before tuberculosis diagnosis, while changes in myeloid inflammation, lymphoid, monocyte and neutrophil responses occurred more proximally to tuberculosis disease. Analysis of gene expression in purified T cells revealed early suppression of Th17 responses in progressors. This was confirmed in an adult BCG re-vaccination cohort, where expression of interferon response genes in blood was associated with suppression of IL-17 expression by BCG-specific CD4 T cells. We concluded that sequential inflammatory dynamics and immune alteration precede tuberculosis disease manifestations, with important implications for developing diagnostics, vaccines and host-directed therapies for tuberculosis.

CD4(+) T cells are critical for the elimination of an immense array of microbial pathogens. Although there are aspects of helper T-cell differentiation that can be modeled as a classic cell-fate commitment, CD4(+) T cells also maintain considerable flexibility in their transcriptional program. Here, we present an overview of chromatin biology during cellular reprogramming and, within this context, envision how the scope of cellular reprogramming may be expanded to further our understanding of the controversy surrounding CD4(+) T lymphocyte plasticity or determinism.

Testing new drugs is critical to improving the treatment of tuberculosis. Quantitative cultures of Mycobacterium tuberculosis on solid media have been used in Phase 1 and 2 trials, but are time and resource intensive. Time to detection (TTD) of growth of M. tuberculosis in automated liquid culture systems is an alternative. TTD has been shown to correlate with CFU in quantitative cultures, and is faster and simpler to perform. We compared TTD in the BACTEC 460 liquid culture system with CFU in a clinical trial that included 110 subjects. Comparing all sputum cultures collected between baseline and 2 months we found a strong negative correlation between log(10) CFU and TTD (rho = -0.91). In addition, when TTD at baseline was compared with 1 and 2 month sputum culture positivity, subjects whose cultures were negative after 1 and 2 months had a significantly longer median baseline TTD compared with subjects whose cultures were positive at 1 and 2 months (5 vs. 3 days and 3 vs. 2 days, respectively). TTD compares closely with CFU and represents a faster, simpler alternative to quantitative cultures.

Global tuberculosis (TB) control may require mass vaccination with a new TB vaccine, such as a recombinant bacille Calmette Guerin (BCG) or attenuated Mycobacterium tuberculosis (MTB). The safety profile of live mycobacterial vaccines in latently infected adults with prior infant BCG vaccination is unknown.Evaluate safety and reactogenicity of BCG revaccination, with or without isoniazid (INH) pretreatment, in adults with latent MTB infection (LTBI).Eighty-two healthy, HIV uninfected, South African adults, with a BCG scar and tuberculin skin test (TST) diameter ≥ 15 mm, were randomized to receive 6 months of INH, starting either before, or 6 months after, intradermal revaccination with BCG Vaccine SSI (Statens Serum Institut, Copenhagen). Safety and reactogenicity data are reported through 3 months post BCG revaccination.Baseline characteristics were similar between treatment arms. Mean baseline TST diameter was 20 ± 4 mm. Seventy-two subjects received BCG revaccination. Injection site erythema (68%) and induration (86%) peaked 1 week after revaccination. Ulceration (76%) peaked at 2 weeks, and resolved by 3 months in all but 3 subjects. Diameter of ulceration was10mm in only 8%, but a residual scar was common (85%). No regional lymphadenitis or serious morbidity related to BCG was seen. Reactogenicity was not affected by INH pretreatment.BCG revaccination of MTB infected adults is safe, well tolerated, and reactogenicity is similar to that of primary BCG vaccination. Clinical trials of live recombinant BCG or attenuated MTB vaccines may be considered in latently infected adults, with or without INH pretreatment (ClinicalTrials.gov identifier: NCT01119521).

Summary Background New drug regimens of greater efficacy and shorter duration are needed for tuberculosis (TB) treatment. The identification of accurate, quantitative, non-culture based markers of treatment response would improve the efficiency of Phase 2 TB drug testing. Methods In an unbiased biomarker discovery approach, we applied a highly multiplexed, aptamer-based, proteomic technology to analyze serum samples collected at baseline and after 8 weeks of treatment from 39 patients with pulmonary TB from Kampala, Uganda enrolled in a Centers for Disease Control and Prevention (CDC) TB Trials Consortium Phase 2B treatment trial. Results We identified protein expression differences associated with 8-week culture status, including Coagulation Factor V, SAA, XPNPEP1, PSME1, IL-11 Rα, HSP70, Galectin-8, α2-Antiplasmin, ECM1, YES, IGFBP-1, CATZ, BGN, LYNB, and IL-7. Markers noted to have differential changes between responders and slow-responders included nectin-like protein 2, EphA1 (Ephrin type-A receptor 1), gp130, CNDP1, TGF-b RIII, MRC2, ADAM9, and CDON. A logistic regression model combining markers associated with 8-week culture status revealed an ROC curve with AUC = 0.96, sensitivity = 0.95 and specificity = 0.90. Additional markers showed differential changes between responders and slow-responders (nectin-like protein), or correlated with time-to-culture-conversion (KLRK1). Conclusions Serum proteins involved in the coagulation cascade, neutrophil activity, immunity, inflammation, and tissue remodeling were found to be associated with TB treatment response. A quantitative, non-culture based, five-marker signature predictive of 8-week culture status was identified in this pilot study.

Background: Treatment of latent infection is needed to protect HIV-infected individuals against tuberculosis. A previous report addressed short-term efficacy of three regimens in HIV-infected adults. We now report on long-term efficacy of the study regimens. Methods: Three daily self-administered regimens were compared in a randomized placebo-controlled trial in 2736 purified protein derivative (PPD)-positive and anergic HIV-infected adults. PPD-positive subjects were treated with isoniazid (INH) for 6 months (6H), INH plus rifampicin for 3 months (3HR), INH plus rifampicin and pyrazinamide for 3 months (3HRZ), or placebo for 6 months. Anergic subjects were randomized to 6H or placebo. Results: 6H initially protected against tuberculosis in PPD-positive individuals; however, benefit was lost within the first year of treatment. Sustained benefit was observed in persons receiving 3HR and 3HRZ. In a Cox regression analysis, the adjusted relative risk for tuberculosis compared with placebo was 0.67 [95% confidence interval (CI), 0.42‐1.07] for 6H, 0.49 (95% CI, 0.29‐0.82) for 3HR, and 0.41 (95% CI, 0.22-0.76) for 3HRZ. When the rifampicin-containing regimens were combined, the adjusted relative risk for tuberculosis compared with placebo was 0.46 (95% CI, 0.29‐0.71). Among anergic subjects, a modest degree of protection with 6H was present (adjusted relative risk, 0.61; 95% CI, 0.32‐1.16). Treatment of latent tuberculosis infection had no effect on mortality. Conclusion: Six months of INH provided short-term protection against tuberculosis in PPD-positive HIV-infected adults. Three month regimens including INH plus rifampicin or INH, rifampicin and pyrazinamide provided sustained protection for up to 3 years.

This article reviews the significant advances in the past year in the basic and clinical aspects of adult tuberculosis (TB). Further research has deepened our understanding of host susceptibility and resistance mechanisms, including cytotoxicity, apoptosis, and antimicrobial polypeptides such as granulysin. Studies have confirmed the effects of HIV infection on risk of disease and disease manifestations, and have defined the effects of HIV on TB transmission. Recent studies also indicate a possible role for extended treatment of active disease and latent infection in HIV-1 infected individuals. Multidrug-resistant disease has been reported on every continent; rapid molecular approaches to the simultaneous diagnosis of TB and detection of rifampin resistance may facilitate prompt initiation of treatment. TB remains one of the major problems in global health.

Pleural tuberculosis (TB) was employed as a model to study T cell apoptosis at sites of active Mycobacterium tuberculosis (MTB) infection in human immunodeficiency virus (HIV)-coinfected (HIV/TB) patients and patients infected with TB alone. Apoptosis in blood and in pleural fluid mononuclear cells and cytokine immunoreactivities in plasma and in pleural fluid were evaluated. T cells were expanded at the site of MTB infection, irrespective of HIV status. Apoptosis of CD4 and non-CD4 T cells in the pleural space occurred in both HIV/TB and TB. Interferon (IFN)-gamma levels were increased in pleural fluid, compared with plasma. Spontaneous apoptosis correlated with specific loss of MTB-reactive, IFN-gamma-producing pleural T cells. Immunoreactivities of molecules potentially involved in apoptosis, such as tumor necrosis factor-alpha, Fas-ligand, and Fas, were increased in pleural fluid, compared with plasma. These data suggest that continued exposure of immunoreactive cells to MTB at sites of infection may initiate a vicious cycle in which immune activation and loss of antigen-responsive T cells occur concomitantly, thus favoring persistence of MTB infection.

SUMMARYImmune parameters were compared in four groups of Ugandan subjects: HIV−and HIV+ adult patients with active pulmonary TB (HIV− PTB n = 38; HIV+ PTB n = 28), patients with HIV infection only (n = 26) and PPD+ healthy controls (n = 25). Compared with healthy controls, CD4 and CD8 T cells from patients with HIV and/or PTB expressed more activation markers (HLA-DR, CD38); their CD8 T cells expressed more CD95 (pre-apoptosis) and less CD28 (co-stimulatory receptor). Peripheral blood mononuclear cells (PBMC) of patients with either HIV or PTB were impaired in interferon-gamma (IFN-γ) production upon antigenic stimulation. PTB (with or without HIV) was characterized by monocytosis, granulocytosis, increased transforming growth factor-beta 1 production and PPD-induced apoptosis. In vivo CD4 T cell depletion, in vitro increased spontaneous CD4 T cell apoptosis and defects in IFN-γ responses upon mitogenic stimulation were restricted to HIV+ subjects (with or without PTB). Overlapping and distinctive immune alterations, associated with PTB and HIV, might explain mutual unfavourable influences of both diseases.

Retrospective cohort studies of tuberculosis suggest that active tuberculosis accelerates the progression of HIV infection. The validity of these findings has been questioned because of their retrospective design, diverse study populations, variable compliance with anti-tuberculous therapy and use of anti-retroviral medication. To assess the impact of tuberculosis on survival in HIV infection we performed a prospective study among HIV-infected Ugandan adults with and without tuberculosis.In a prospective cohort study, 230 patients with HIV-associated tuberculosis and 442 HIV-infected subjects without tuberculosis were followed for a mean duration of 19 months for survival. To assess changes in viral load over 1 year, 20 pairs of tuberculosis cases and controls were selected and matched according to baseline CD4 lymphocyte count, age, sex and tuberculin skin test status.During the follow-up period, 63 out of of 230 tuberculosis cases (28%) died compared with 85 out of 442 controls (19%), with a crude risk ratio of 1.4 [95% confidence interval (CI), 1.07-1.87]. Most deaths occurred in patients with CD4 lymphocyte counts200 x 10(6) cells/l at baseline (n = 99) and occurred with similar frequency in the tuberculosis cases (46%) and the controls (44%). When the CD4 lymphocyte count was200 x 10(6)/l, however, the relative risk of death in HIV-associated tuberculosis was 2.1 (95% CI, 1.27-3.62) compared with subjects without tuberculosis. For subjects with a CD4 lymphocyte count200 x 10(6)/l, the 1-year survival proportion was slightly lower in the cases than in the controls (0.91 versus 0.96), but by 2 years the survival proportion was significantly lower in the cases than in the controls (0.84 versus 0.91; P0.02; log-rank test). For subjects with a CD4 lymphocyte count of 200 x 10(6) cells/l or fewer, the survival proportion at 1 year for the controls was lower than cases (0.59 versus 0.64), but this difference was not statistically significant (P = 0.53; logrank test). After adjusting for age, sex, tuberculin skin test status, CD4 lymphocyte count, and history of HIV-related infections, the overall relative hazard for death associated with tuberculosis was 1.81 (95% CI, 1.24-2.65). In a nested Cox regression model, the relative hazard for death was 3.0 (95% CI, 1.62-5.63) for subjects with CD4 lymphocyte counts200 x 10(6)/l and 1.5 (95% CI, 0.99-2.40) for subjects with a CD4 lymphocyte count of 200 x 10(6)/l or fewer.The findings from this prospective study indicate that active tuberculosis exerts its greatest effect on survival in the early stages of HIV infection, when there is a reserve capacity of the host immune response. These observations provide a theoretical basis for the treatment of latent tuberculous infection in HIV-infected persons.

Three herpesviruses--herpes simplex, varicella-zoster, and cytomegalovirus--commonly cause respiratory tract infections in immunocompromised patients. Adenoviruses and measles virus are also significant causes of respiratory disease in this population. Diagnosis of herpesvirus infections is difficult because these viruses can establish latency and are often shed intermittently in the absence of invasive disease. A positive respiratory tract culture of herpesviruses alone is not diagnostic of active invasive disease. Preventive measures should focus on limiting the patient's exposure to active infection, broad use of available vaccines in children and susceptible adults, and use of hyperimmune globulin and chemoprophylaxis in high-risk patients. Adenovirus pneumonia is diagnosed by viral culture and rapid antigen detection assays, whereas measles pneumonia is often identifiable by the characteristic rash. Treatment of either adenovirus or measles pneumonia is primarily supportive.

Despite enhanced laboratory techniques such as viral culture, rapid antigen detection, and gene amplification, a confident diagnosis of viral pneumonia continues to be a challenge. The nonspecific nature of clinical characteristics and the extreme sensitivity of laboratory techniques make the diagnosis difficult, even when a viral agent is detected. Understanding the limitations of these technological advances and the use of histopathologic techniques can greatly enhance a skilled clinician's ability to make an accurate diagnosis.

Effective treatment regimens for pulmonary tuberculosis are difficult to assess because of the slow growth rate of Mycobacterium tuberculosis in culture and its protracted clearance from sputum. A rapid method that reflects effective antimicrobial activity would markedly advance evaluation of treatment and promote the assessment of new antituberculosis drugs. Conventional methods measure the progressive reduction of numbers of acid-fast bacilli in the sputum smear and the clearance of organisms in sputum culture. In this study, we measured levels of M. tuberculosis 85B (alpha antigen) messenger RNA (mRNA), 16S ribosomal RNA (rRNA), and IS6110 DNA in patients' sputa to ascertain whether they could serve as potential surrogate markers of response to chemotherapy. Sputum specimens were sequentially collected for up to a year from 19 smear-positive pulmonary tuberculosis patients receiving an optimal drug treatment regimen. Nucleic acids were isolated from these specimens, and two M. tuberculosis molecular targets (mRNA, DNA) were quantified, using the ABI Prism 7700 Sequence Detection System. The Mycobacterium genus-specific 16S rRNA was quantified with a limiting dilution RT-PCR assay. Results show that levels of 85B mRNA declined after initiation of therapy, as did viable M. tuberculosis colony counts, with 90% of patients becoming negative for both markers after 2 mo of treatment. The rapid disappearance of M. tuberculosis mRNA from sputum suggests that it is a good indicator of microbial viability and a useful marker for rapid assessment of response to chemotherapy.

Mycobacterium tuberculosis (MTB)-induced T cell responses are depressed in peripheral blood mononuclear cells of persons with newly diagnosed pulmonary tuberculosis (TB), and levels of interferon (IFN)-gamma remain low even after completion of antituberculous therapy. Loss of MTB-reactive T cells through apoptotic mechanisms could account for this prolonged T cell hyporesponsiveness. T cell apoptosis was studied in TB patients and healthy control subjects. Both spontaneous and MTB-induced apoptosis (in CD4 and non-CD4 T cells) from TB patients was increased when compared with healthy control subjects, whereas coculture with control antigen (candida) had no effect on T cell apoptosis in either group of study subjects. An inverse correlation existed between increased MTB-induced T cell apoptosis and IFN-gamma and interleukin (IL)-2 immunoreactivities. Successful antituberculous chemotherapy resulted in a 50% reduction in both spontaneous and MTB-induced apoptosis, which coincided with 3- and 8-fold increases in levels of MTB-stimulated IL-2 and IFN-gamma, respectively. These data indicate that apoptotic pathways are operant during active MTB infection and may contribute to deletion of MTB-reactive T cells and the immunopathogenesis of this disease.

We present a case of paradoxical clinical deterioration during antituberculosis therapy in an HIV-infected adult with pulmonary TB. The clinical course was characterized by marked cervical and mediastinal adenopathy accompanied by fever and weight loss during simultaneous treatment of TB and HIV disease. After extensive investigation for causes of therapeutic failure, the paradoxical reaction was attributed to partial immune reconstitution related to highly effective antiretroviral therapy. Due to the high prevalence of TB in HIV-infected patients, it is important to recognize this phenomenon and understand that it is usually self-limited. (CHEST 1998; 114:933–936)