Tuberculosis-specific T-cell Research Articles

Brain abscess caused by Mycobacterium tuberculosis in an immunocompetent patient: a case report and literature review.

Brain abscesses caused by Mycobacterium tuberculosis are rarely reported and are typically found in immunocompromised patients. The challenges in identifying the pathogen and the frequent delays in initiating appropriate therapy often result in a poor prognosis for patients. We herein report a rare case of a brain abscess caused by M. tuberculosis in a 63-year-old patient with a history of hypertension but no history of pulmonary tuberculosis. The patient was hospitalized twice because of worsening right upper limb weakness and speech difficulties. Both a pus culture and cerebrospinal fluid culture were negative. Ultimately, the diagnosis of M. tuberculosis infection was confirmed through next-generation sequencing and the tuberculosis-specific T-cell test (T-SPOT). The patient's symptoms improved following anti-tuberculosis treatment. This case highlights the importance of considering M. tuberculosis as a potential cause of brain abscesses, even in immunocompetent individuals, and underscores the need for early diagnosis for effective treatment.

Impaired Mycobacterium tuberculosis-specific T-cell memory phenotypes and functional profiles among adults with type 2 diabetes mellitus in Uganda.

Efforts to eradicate tuberculosis (TB) are threatened by diabetes mellitus (DM), which confers a 3-fold increase in the risk of TB disease. The changes in the memory phenotypes and functional profiles of Mycobacterium tuberculosis (Mtb)-specific T cells in latent TB infection (LTBI)-DM participants remain poorly characterised. We, therefore, assessed the effect of DM on T-cell phenotype and function in LTBI and DM clinical groups. We compared the memory phenotypes and function profiles of Mtb-specific CD4+ and CD8+ T cells among participants with LTBI-DM (n=21), LTBI-only (n=17) and DM-only (n=16). Peripheral blood mononuclear cells (PBMCs) were stimulated with early secretory antigenic 6 kDa (ESAT-6) and culture filtrate protein 10 (CFP-10) peptide pools or phytohemagglutinin (PHA). The memory phenotypes (CCR7/CD45RA), and functional profiles (HLA-DR, PD-1, CD107a, IFN-γ, IL-2, TNF, IL-13, IL-17A) of Mtb-specific CD4+ and CD8+ T cells were characterised by flow cytometry. Naïve CD4+ T cells were significantly decreased in the LTBI-DM compared to the LTBI-only participants [0.47 (0.34-0.69) vs 0.91 (0.59-1.05); (p<0.001)]. Similarly, CD8+ HLA-DR expression was significantly decreased in LTBI-DM compared to LTBI-only participants [0.26 (0.19-0.33) vs 0.52 (0.40-0.64); (p<0.0001)], whereas CD4+ and CD8+ PD-1 expression was significantly upregulated in the LTBI-DM compared to the LTBI-only participants [0.61 (0.53-0.77) vs 0.19 (0.10-0.28); (p<0.0001) and 0.41 (0.37-0.56) vs 0.29 (0.17-0.42); (p=0.007)] respectively. CD4+ and CD8+ IFN-γ production was significantly decreased in the LTBI-DM compared to the LTBI-only participants [0.28 (0.19-0.38) vs 0.39 (0.25-0.53); (p=0.030) and 0.36 (0.27-0.49) vs 0.55 (0.41-0.88); (p=0.016)] respectively. CD4+ TNF and CD8+ IL-17A production were significantly decreased in participants with LTBI-DM compared to those with LTBI-only [0.38 (0.33-0.50) vs 0.62 (0.46-0.87); (p=0.004) and 0.29 (0.16-0.42) vs 0.47 (0.29-0.52); (0.017)] respectively. LTBI-DM participants had significantly lower dual-functional (IFN-γ+IL-2+ and IL-2+TNF+) and mono-functional (IFN-γ+ and TNF+) CD4+ responses than LTBI-only participants. LTBI-DM participants had significantly decreased dual-functional (IFN-γ+IL-2+, IFN-γ+ TNF+ and IL-2+TNF+) and mono-functional (IFN-γ+, IL-2+ and TNF+) central and effector memory CD4+ responses compared to LTBI-only participants. Type 2 DM impairs the memory phenotypes and functional profiles of Mtb-specific CD4+ and CD8+ T cells, potentially indicating underlying immunopathology towards increased active TB disease risk.

BCG-Vaccinated Children with Contact to Tuberculosis Patients Show Delayed Conversion of Mycobacterium tuberculosis-Specific IFN-γ Release.

Mycobacterium (M.) bovis BCG vaccination is recommended for healthy babies after birth in several countries with a high prevalence of tuberculosis, including Ghana. Previous studies showed that BCG vaccination prevents individuals from developing severe clinical manifestations of tuberculosis, but BCG vaccination effects on the induction of IFN-γ after M. tuberculosis infection have hardly been investigated. Here, we performed IFN-γ-based T-cell assays (i.e., IFN-γ Release Assay, IGRA; T-cell activation and maturation marker assay, TAM-TB) in children who had contact with index tuberculosis patients (contacts). These contacts were classified as either being BCG vaccinated at birth (n = 77) or non-BCG-vaccinated (n = 17) and were followed up at three timepoints for a period of one year to determine immune conversion after M. tuberculosis exposure and potential infection. At baseline and month 3, BCG-vaccinated contacts had significantly lower IFN-γ levels after stimulation with M. tuberculosis-specific proteins as compared to non-BCG-vaccinated contacts. This resulted in decreased proportions of positive IGRA results (BCG-vaccinated: 60% at baseline, 57% at month 3; non-BCG-vaccinated: 77% and 88%, respectively) at month 3. However, until month 12, immune conversion in BCG-vaccinated contacts led to balanced proportions in IGRA responders and IFN-γ expression between the study groups. TAM-TB assay analyses confirmed higher proportions of IFN-γ-positive T-cells in non-BCG-vaccinated contacts. Low proportions of CD38-positive M. tuberculosis-specific T-cells were only detected in non-BCG-vaccinated contacts at baseline. These results suggest that BCG vaccination causes delayed immune conversion as well as differences in the phenotype of M. tuberculosis-specific T-cells in BCG-vaccinated contacts of tuberculosis patients. These differences are immune biomarker candidates for protection against the development of severe clinical tuberculosis manifestations.

Mycobacterium tuberculosis-Specific T-Cell Responses Are Impaired During Late Pregnancy With Elevated Biomarkers of Tuberculosis Risk Postpartum.

Pregnancy is a risk factor for progression from latent tuberculosis infection to symptomatic tuberculosis. However, how pregnancy influences T-cell responses to Mycobacterium tuberculosis is unknown. We measured M. tuberculosis-specific cytokines, T-cell memory markers, and overall CD4+ and CD8+ T-cell activation by flow cytometry from 49 women (18 with and 31 without HIV) who became pregnant while enrolled in a randomized controlled trial of preexposure prophylaxis for HIV. We analyzed data using COMPASS, an established statistical method for evaluating overall antigen-specific T-cell responses. Pregnant women with latent tuberculosis infection demonstrated significantly diminished M. tuberculosis-specific CD4+ cytokine responses in the third trimester (COMPASS polyfunctional score [PFS], 0.07) compared before (PFS, 0.15), during (PFS, 0.13 and 0.16), and after pregnancy (PFS, 0.14; P = .0084, Kruskal-Wallis test). Paradoxically, M. tuberculosis-specific CD8+ cytokines and nonspecifically activated T-cells increased during late pregnancy. Nonspecific T-cell activation, a validated biomarker for progression from latent tuberculosis infection to tuberculosis disease, increased in latent tuberculosis infection-positive women postpartum, compared with latent tuberculosis infection-negative women. Pregnancy-related functional T-cell changes were most pronounced during late pregnancy. Both M. tuberculosis-specific T-cell changes during pregnancy and increases in immune activation postpartum may contribute to increased risk for tuberculosis progression. NCT0557245.

Computational discovery and ex-vivo validation study of novel antigenic vaccine candidates against tuberculosis

Tuberculosis (TB) is a complex infectious bacterial disease, which has evolved with highly successful mechanisms to interfere with host defenses and existing classes of antibiotics to resist eradication. The single obtainable TB vaccine, Bacille Calmette-Guerin (BCG) has failed to provide regular defense for respiratory TB in adults. In this study, a bioinformatics and immunoinformatics approach was applied on Mycobacterium tuberculosis (Mtb) H37Rv proteomes to discover the potential subunit vaccine candidates that elicit both tuberculosis-specific T-cells and B-cell immune response. A total of 4049 proteins of MtbH37RvMtbH37Rv were retrieved and subjected to in silico sequence-based analysis. Finally, five (P9WL69 (Rv2599), P9WIG1 (Rv0747), P9WLQ1 (Rv1987), O53608 (Rv0063), O06624 (Rv1566c)) novel putative proteins were selected. Among the five putative antigenic vaccine candidates, P9WL69 protein was selected for the ex-vivo validation study. The P9WL69 protein encoding gene was amplified and cloned on pET21b vector. The success of the recombinant clone (pET21b-RV2599) was confirmed by colony PCR, insert release test and sequencing. Furthermore, the identified epitopes of the P9WL69 protein were considered for in silico docking and molecular dynamics simulation study using Toll-like Receptors (TLRs) (TLR-2, TLR-4, TLR-9), Mannose receptor, and Myeloid differentiation 88 (MYD88) to understand their binding affinity towards the development of immunogenic vaccines against tuberculosis.

Impact of antiretroviral and tuberculosis therapies on CD4+ and CD8+ HIV/M. tuberculosis-specific T-cell in co-infected subjects

BackgroundHuman Immunodeficiency Virus (HIV) infection is a risk factor for tuberculosis (TB). Antiretroviral therapy (ART) changed HIV clinical management but it is still unclear how pre-existing HIV/Mycobacterium tuberculosis (Mtb)-specific CD4+ and CD8+ T-cells are restored. Aimto evaluate the impact of ART and TB therapies on the functional and phenotypic profile of Mtb-specific antigen-response of CD4+ and CD8+ T-cells in prospectively enrolled HIV-TB co-infected patients. MethodsART-naïve HIV-infected patients, with or without active TB or latent TB infection (LTBI), were enrolled before and after starting ART and TB therapies. Peripheral blood mononuclear cells (PBMC) were stimulated overnight with Mtb and HIV antigens (GAG). Cytokine expression and phenotype profile were evaluated by flow cytometry. Cytomegalovirus (CMV) and staphylococcal enterotoxin B (SEB) were also used. ResultsThe median of absolute number of CD4+ T-cells increased after ART and TB therapies in all groups analyzed, while the median of absolute number of CD8+ T-cells decreases in HIV and HIV-LTBI groups. Treatments significantly increased the frequency of Mtb-specific CD4+ T-cells in the HIV-LTBI (p = 0.015) with a rise of the central memory compartment. The magnitude of the CD4+ T-cell response to HIV-GAG significantly increased in active TB (p = 0.03), whereas the magnitude of CMV-specific CD4+ T-cell response decreased in all the groups. Similarly, the treatments increased the number of Mtb-specific CD8+ responders in both HIV-LTBI and HIV-TB groups, whereas the phenotype distribution was dependent on the antigens used and on the stage of infection/disease. ConclusionsAfter therapies the median of absolute number and the proportion of CD4+ T-cells increased in all groups whereas the median of absolute count and proportion of CD8+ T-cells decreased in the HIV and HIV-LTBI subjects. Interestingly, an increased frequency of CD4+ T-cell response to RD1 proteins in HIV-LTBI subjects was found. These results contribute to a better understanding of the effect of ART and TB therapies on the modulation of Mtb-specific CD4+ and CD8+ T-cells subsets.

Constitutive STAT3 phosphorylation and IL-6/IL-10 co-expression are associated with impaired T-cell function in tuberculosis patients.

T-cells critically contribute to protection against Mycobacterium tuberculosis infection, and impaired T-cell responses can lead to disease progression. Pro-inflammatory and immunosuppressive cytokines affect T-cells, and fine-tuned regulation of cytokine signaling via the Jak/STAT signaling pathways is crucial for appropriate T-cell function. Constitutive STAT3 phosphorylation as a consequence of aberrant cytokine signaling has been described to occur in pathognomonic T-cell responses in inflammatory and autoimmune diseases. We characterized blood samples from tuberculosis patients (n=28) and healthy contacts (n=28) from Ghana for M. tuberculosis-specific T-cell responses, constitutive cytokine production, and SOCS3 and pSTAT3 expression. Lentiviral modulation of primary CD4+ T-cells was performed to determine the effects of SOCS3 on T-cell functions. T-cells from tuberculosis patients expressed higher levels of IL-10 and IL-6 and lower levels of T helper type (TH)17 cytokines after M. tuberculosis-specific stimulation compared to healthy contacts. In addition, tuberculosis patients had higher IL-10 and IL-6 levels in the supernatants of non-stimulated immune cells and plasma samples compared to healthy contacts. Notably, aberrant cytokine expression was accompanied by high constitutive pSTAT3 levels and SOCS3 expression in T-cells. Multivariate analysis identified an IL-6/IL-10 co-expression-based principal component in tuberculosis patients that correlated with high pSTAT3 levels. SOCS3 contributed to a regulatory component, and tuberculosis patients with high SOCS3 expression showed decreased TH1 cytokine expression and impaired IL-2-induced STAT5 phosphorylation. SOCS3 over-expression in primary CD4+ T-cells confirmed the SOCS3 inhibitory function on IL-2-induced STAT5 phosphorylation. We conclude that constitutive pSTAT3 and high SOCS3 expression are influential factors that indicate impaired T-cell functions in tuberculosis patients.

Novel T-cell assays for the discrimination of active and latent tuberculosis infection: the diagnostic value of PPE family.

The diagnosis of active and latent tuberculosis remains a challenge. Although a new approach based on detecting Mycobacterium tuberculosis-specific T-cells has been introduced, it cannot distinguish between latent infection and active disease. The aim of this study was to evaluate the diagnostic potential of interleukin-2 (IL-2) as biomarker after specific antigen stimulation with PE35 and PPE68 for the discrimination of active and latent tuberculosis infection (LTBI). The production of IL-2 was measured in the antigen-stimulated whole-blood supernatants following stimulation withrecombinant PE35 and PPE68. The discrimination performance (assessed by the area under ROC curve) for IL-2 following stimulation withrecombinant PE35 and PPE68 between LTBI and patients with active TB were 0.837 [95% confidence interval (CI) 0.72-0.97] for LTBI diagnosis and 0.75 (95% CI 0.63-0.89) for active TB diagnosis, respectively. Applying the 6.4pg/mL cut-off for IL-2 induced by PE35 in the present study population resulted in sensitivity of 78%, specificity of 83%, PPV of 83% and NPV of 78% for the discrimination of active TB and LTBI. In addition, a sensitivity of 81%, specificity of 71%, PPV of 68 and 83% of NPV was reported based on the 4.4pg/mL cut-off for IL-2 induced by PPE68. This study confirms IL-2 induced by PE35 and PPE68 as a sensitive and specific biomarker and highlights IL-2 as new promising adjunct markers for discriminating of LTBI and active TB disease.

T cells and adaptive immunity to Mycobacterium tuberculosis in humans.

The adaptive immune response mediated by T cells is critical for control of Mycobacterium tuberculosis (M. tuberculosis) infection in humans. However, the M. tuberculosis antigens and host T-cell responses that are required for an effective adaptive immune response to M. tuberculosis infection are yet to be defined. Here, we review recent findings on CD4(+) and CD8(+) T-cell responses to M. tuberculosis infection and examine the roles of distinct M. tuberculosis-specific T-cell subsets in control of de novo and latent M. tuberculosis infection, and in the evolution of T-cell immunity to M. tuberculosis in response to tuberculosis treatment. In addition, we discuss recent studies that elucidate aspects of M. tuberculosis-specific adaptive immunity during human immunodeficiency virus co-infection and summarize recent findings from vaccine trials that provide insight into effective adaptive immune responses to M. tuberculosis infection.

A novel liposomal adjuvant system, CAF01, promotes long-lived Mycobacterium tuberculosis-specific T-cell responses in human

Here, we report on a first-in-man trial where the tuberculosis (TB) vaccine Ag85B-ESAT-6 (H1) was adjuvanted with escalating doses of a novel liposome adjuvant CAF01. On their own, protein antigens cannot sufficiently induce immune responses in humans, and require the addition of an adjuvant system to ensure appropriate delivery and concomitant immune activation. To date no approved adjuvants are available for induction of cellular immunity, which seems essential for a number of vaccines, including vaccines against TB. We vaccinated four groups of human volunteers: a non-adjuvanted H1 group, followed by three groups with escalating doses of CAF01-adjuvanted H1 vaccine. All subjects were vaccinated at 0 and 8 weeks and followed up for 150 weeks. Vaccination did not cause local or systemic adverse effects besides transient soreness at the injection site. Two vaccinations elicited strong antigen-specific T-cell responses which persisted after 150 weeks follow-up, indicating the induction of a long-lasting memory response in the vaccine recipients. These results show that CAF01 is a safe and tolerable, Th1-inducing adjuvant for human TB vaccination trials and for vaccination studies in general where cellular immunity is required.

Programmed Death 1 Lives Up to Its Reputation in Active Tuberculosis

Programmed death 1 (PD-1; CD279), also known as programmed cell death protein 1 (PDCD-1), is a cell surface receptor of the immunoglobulin superfamily found on immune effector cells. It belongs to the extended CD28/CTLA-4 family of T-cell regulators and is expressed by a range of immune cells, including B cells, natural killer cells, and monocytes. Binding of PD-1 to one of its ligands, PD ligand 1 (PD-L1) or PD-L2, on antigen-presenting cells (APCs) is known to negatively regulate T-cell receptor signaling and inhibit T-cell activation [1]. In this issue of the Journal, Singh et al [2] demonstrate that the coinhibitory receptor PD-1 and the PD ligands are expressed on higher percentages of peripheral blood mononuclear cells (PBMCs) of patients with active tuberculosis than those of healthy controls in an area of tuberculosis endemicity. The expression of PD-1 on T cells is upregulated following in vitro stimulation with Mycobacterium tuberculosis antigens, and the PD-1 blockade in vitro enhances interferon γ (IFN-γ) and interleukin 2 production by specific T cells and rescues them from undergoing apoptosis. Of particular significance, the authors demonstrate during a 1-year follow-up period a significant decrease in the frequency of PD-1–expressing T cells after successful antituberculosis treatment that led to restoration of the M. tuberculosis–specific T-cell cytokine response in vitro. These data substantiate the role of PD-1–PD-L pathways in the inhibition of T-cell responses in patients with active tuberculosis. Optimal T-cell activation during infection requires 2 signals. The first signal is generated by T-cell–receptor recognition ofpeptide–majorhistocompatibilitycom

CD8+T Cells Provide an Immunologic Signature of Tuberculosis in Young Children

The immunologic events surrounding primary Mycobacterium tuberculosis infection and development of tuberculosis remain controversial. Young children who develop tuberculosis do so quickly after first exposure, thus permitting study of immune response to primary infection and disease. We hypothesized that M. tuberculosis-specific CD8(+) T cells are generated in response to high bacillary loads occurring during tuberculosis. To determine if M. tuberculosis-specific T cells are generated among healthy children exposed to M. tuberculosis and children with tuberculosis. Enzyme-linked immunosorbent spot assays were used to measure IFN-γ production in response to M. tuberculosis-specific proteins ESAT-6/CFP-10 by peripheral blood mononuclear cells and CD8(+) T cells isolated from Ugandan children hospitalized with tuberculosis (n = 96) or healthy tuberculosis contacts (n = 62). The proportion of positive CD8(+) T-cell assays and magnitude of CD8(+) T-cell responses were significantly greater among young (<5 yr) tuberculosis cases compared with young contacts (P = 0.02, Fisher exact test, P = 0.01, Wilcoxon rank-sum, respectively). M. tuberculosis-specific T-cell responses measured in peripheral blood mononuclear cells were equivalent between groups. Among young children, M. tuberculosis-specific CD8(+) T cells develop in response to high bacillary loads, as occurs during tuberculosis, and are unlikely to be found after M. tuberculosis exposure. T-cell responses measured in peripheral blood mononuclear cells are generated after M. tuberculosis exposure alone, and thus cannot distinguish exposure from disease. In young children, IFN-γ-producing M. tuberculosis-specific CD8(+) T cells provide an immunologic signature of primary M. tuberculosis infection resulting in disease.

Tuberculosis-specific T-cell response after recent treatment and remote cure

To the Editors: Tuberculosis (TB) remains a major global health problem. A major factor contributing to the current TB epidemic is the resistance of Mycobaterium tuberculosis to first-line anti-TB treatment. Therefore, there is a need for an appropriate and reliable tool to evaluate treatment efficacy. Several studies using the T-SPOT®. TB assay (Oxford Immunotec Ltd, Oxford, UK) reported a decline in the number of spots after successful anti-TB treatment, particularly in the 6-kDa early secretory antigenic target (ESAT-6) panel [1–3]; however, to our knowledge, no data have been published on cured TB patients who contracted TB before isoniazid and rifampicin became available. Therefore, the naturally long-term range of ESAT-6- and culture filtrate protein (CFP)-10-specific T-cell responses in patients with TB who did not receive modern anti-TB treatment is not known. Because most patients in prior studies were born in high-burden, TB-endemic countries, the T-cell kinetics might be influenced by reinfection. Therefore, the aim of this report is to compare the ESAT-6- and CFP-10-specific T-cell responses in a low-burden country with a low risk of reinfection between patients with a history of active TB before 1955, and patients with recently treated active TB. Venous blood for T-SPOT®. TB was collected from 24 outpatient subjects with a history of active TB before 1955 (group 1), obtained by clinical history-taking, patients’ medical records or documentation of a sanatorium admission. 16 of these patients had been admitted to a sanatorium, two patients underwent surgery and six patients were treated at home with para-aminosalicylic acid therapy or bedrest. Furthermore, 27 patients with recent active TB (group 2) were prospectively enrolled between March and May 2008. Active TB was defined as fulfilling one or more of the following criteria: 1) positive culture for M. tuberculosis (n=22); 2) positive PCR for M. …

Cellular Immune Responses to Nine Mycobacterium tuberculosis Vaccine Candidates following Intranasal Vaccination

BackgroundThe identification of Mycobacterium tuberculosis vaccines that elicit a protective immune response in the lungs is important for the development of an effective vaccine against tuberculosis.Methods and Principal FindingsIn this study, a comparison of intranasal (i.n.) and subcutaneous (s.c.) vaccination with the BCG vaccine demonstrated that a single moderate dose delivered intranasally induced a stronger and sustained M. tuberculosis-specific T-cell response in lung parenchyma and cervical lymph nodes of BALB/c mice than vaccine delivered subcutaneously. Both BCG and a multicomponent subunit vaccine composed of nine M. tuberculosis recombinant proteins induced strong antigen-specific T-cell responses in various local and peripheral immune compartments. Among the nine recombinant proteins evaluated, the alanine proline rich antigen (Apa, Rv1860) was highly antigenic following i.n. BCG and immunogenic after vaccination with a combination of the nine recombinant antigens. The Apa-induced responses included induction of both type 1 and type 2 cytokines in the lungs as evaluated by ELISPOT and a multiplexed microsphere-based cytokine immunoassay. Of importance, i.n. subunit vaccination with Apa imparted significant protection in the lungs and spleen of mice against M. tuberculosis challenge. Despite observed differences in the frequencies and location of specific cytokine secreting T cells both BCG vaccination routes afforded comparable levels of protection in our study.Conclusion and SignificanceOverall, our findings support consideration and further evaluation of an intranasally targeted Apa-based vaccine to prevent tuberculosis.

Clinical Significance of Interleukin-2/Gamma Interferon Ratios in Mycobacterium tuberculosis-Specific T-Cell Signatures

The simultaneous determination of interleukin-2 (IL-2) and gamma interferon (IFN-γ) in QuantiFERON-TB test plasma supernatants permitted the detection of shifts in Mycobacterium tuberculosis-specific T-cell signatures. A subset of the 84 subjects tested revealed a significantly elevated IL-2/IFN-γ ratio, which may be a marker for the successful elimination of M. tuberculosis infection.

Mycobacterium tuberculosis impairs dendritic cell response by altering CD1b, DC‐SIGN and MR profile

During a chronic infection such as tuberculosis, the pool of tissue dendritic cells (DC) must be renewed by recruitment of both circulating DC progenitors and monocytes (Mo). However, the microenvironment of the inflammatory site affects Mo differentiation. As DC are critical for initiating a Mycobacterium tuberculosis-specific T-cell response, we argue that interference of M. tuberculosis with a correct DC generation would signify a mechanism of immune evasion. In this study, we showed that early interaction of γ-irradiated M. tuberculosis with Mo subverts DC differentiation in vitro. We found that irradiated M. tuberculosis effect involves (1) the loss of a significant fraction of monocyte population and (2) an altered differentiation process of the surviving monocyte subpopulation. Moreover, in the absence of irradiated M. tuberculosis, DC consist in a major DC-specific intercellular adhesion molecule 3-grabbing non-integrin receptor (DC-SIGN(high))/CD86(low) and minor DC-SIGN(low)/CD86(high) subpopulations, whereas in the presence of bacteria, there is an enrichment of DC-SIGN(low)/CD86(high) population. Besides, this population enlarged by irradiated M. tuberculosis, which is characterized by a reduced CD1b expression, correlates with a reduced induction of specific T-lymphocyte proliferation. The loss of CD1molecules partially involves toll-like receptors (TLR-2)/p38 MAPK activation. Finally, several features of Mo, which have been differentiated into DC in the presence of irradiated M. tuberculosis, resemble the features of DC obtained from patients with active tuberculosis. In conclusion, we suggest that M. tuberculosis escapes from acquired immune response in tuberculosis may be caused by an altered differentiation into DC leading to a poor M. tuberculosis-specific T-cell response.

Local immunodiagnosis of pulmonary tuberculosis by enzyme-linked immunospot

Lymphocytes are crucial in the immune defence against Mycobacterium tuberculosis (MTB) infection. The aim of the present study was to ascertain whether or not MTB-specific lymphocytes are selectively compartmentalised in the lungs of patients with minimal active pulmonary tuberculosis (PTB). Patients with smear-negative MTB-culture-confirmed PTB were prospectively recruited. Differential cell counts, immunophenotyping with monoclonal antibodies directed against the cell surface markers CD4, CD8, CD4CD45RA, CD4CD45R0, CD38, human leukocyte antigen DR, CD19, CD3, CD57 and CD16 and MTB-specific enzyme-linked immunospot assays of peripheral blood mononuclear cells and bronchoalveolar lavage (BAL) mononuclear cells with 6-kDa early secretory antigenic target and culture filtrate protein 10 were performed. Among 12 patients with culture-confirmed smear-negative PTB, no differences were found in the distribution of total CD4 or CD8 T-cells in peripheral blood or BAL fluid (BALF). Activated human leukocyte antigen-DR-positive cells, as well as memory CD4CD45R0-positive T-cells, were expanded among cells of the BALF. Compared with a group of control patients with alternative pulmonary pathologies, there was no significant difference in lymphocyte subpopulations. However, 6-kDa early secretory antigenic target- and culture filtrate protein 10-specific lymphocytes were more concentrated, with a median BALF:peripheral blood ratio of 9.9 and 8.9, respectively, in patients with PTB. Mycobacterium tuberculosis-specific T-cells are highly selectively compartmentalised at the site of infection in active pulmonary tuberculosis.

Human T-cell responses to 25 novel antigens encoded by genes of the dormancy regulon of Mycobacterium tuberculosis

The dormancy (DosR) regulon of Mycobacterium tuberculosis is expressed in vitro during hypoxia and low-dose nitric oxide stimulation. Tubercle bacilli are thought to encounter these conditions in humans during latent infection. In this study, immune responses were evaluated to 25 most strongly induced DosR-regulon-encoded proteins, referred to as latency antigens. Proliferation assays were performed using M. tuberculosis-specific T-cell lines and peripheral blood mononuclear cells (PBMC) from tuberculosis (TB) patients, tuberculin skin test positive (TST+) individuals and uninfected controls. All 25 latency antigens were able to induce production of interferon-gamma (IFN-γ) by T-cell lines. Eighteen latency antigens were also recognized by PBMC of M. tuberculosis-infected individuals, which indicates expression of the DosR-regulon during natural infection. Differential analysis showed that TST+ individuals recognized more latency antigens and with a stronger cumulative IFN-γ response than TB patients, while the opposite profile was found for culture filtrate protein-10. In particular Rv1733c, Rv2029c, Rv2627c and Rv2628 induced strong IFN-γ responses in TST+ individuals, with 61%, 61%, 52% and 35% responders, respectively. In conclusion, several new M. tuberculosis antigens were identified within the DosR-regulon. Particularly strong IFN-γ responses to latency antigens were observed in latently infected individuals, suggesting that immune responses against these antigens may contribute to controlling latent M. tuberculosis infection.

Comparison of the Immunological Activity of Five Defined Antigens from Mycobacterium tuberculosis in Seven Inbred Guinea Pig Strains. The 38‐kDa Antigen is Immunodominant

We have examined the immunological activity of five affinity-purified protein antigens from Mycobacterium tuberculosis in seven inbred and one outbred guinea pig strains. The test systems were measurements of delayed-type hypersensitivity (Dth) responses, lymphocyte stimulation assays (LS), and antibody response measurements. The results showed significant differences in the immunogenicity of the single-protein antigens and, when the antigens were considered separately, highly significant guinea pig strain differences. The outbred guinea pig strain behaved as a Dth high responder to all antigens studied. The order of magnitude of the Dth responses was not usually correlated with that of the corresponding antibody responses for the individual guinea pig strain-antigen combinations. In particular, when compared with the other strains, strain 2 guinea pigs generally gave the lowest Dth, but the highest antibody responses. A 38,000 molecular weight protein, possessing M. tuberculosis complex-specific B-cell determinants, appeared immunodominant in 5 out of 7 strains. Our Dth data in the inbred strains further suggest the presence of an M. tuberculosis-specific T-cell epitope. A T-cell line, 11D9, derived from the high-responder guinea pig strain 13 reactive to this protein, was shown to be able to confer a tuberculin-like skin reaction in vivo. LS assays with recombinant 38-kDa protein and truncated versions of the protein mapped the 11D9-defined T-cell epitope to the middle part of the molecule.