Mycobacterium tuberculosis Immune Response in Patients With Immune-Mediated Inflammatory Disease.

Subjects with immune-mediated inflammatory diseases (IMID), such as rheumatoid arthritis (RA), have an intrinsic higher probability to develop active-tuberculosis (TB) compared to the general population. The risk ranges from 2.0 to 8.9 in RA patients not receiving therapies. According to the WHO, the RA prevalence varies between 0.3% and 1% and is more common in women and in developed countries. Therefore, the identification and treatment of TB infection (TBI) in this fragile population is important to propose the TB preventive therapy. We aimed to study the M. tuberculosis (Mtb) specific T-cell response to find immune biomarkers of Mtb burden or Mtb clearance in patients with different TB status and different risk to develop active-TB disease. We enrolled TBI subjects as example of Mtb-containment, the active-TB as example of a replicating Mtb status, and the TBI-IMID as fragile population. To study the Mtb-specific response in a condition of possible Mtb sterilization, we longitudinally enrolled TBI subjects and active-TB patients before and after TB therapy. Peripheral blood mononuclear cells were stimulated overnight with Mtb peptides contained in TB1- and TB2-tubes of the Quantiferon-Plus kit. Then, we characterized by cytometry the Mtb-specific CD4 and CD8 T cells. In TBI-IMID, the TB therapy did not affect the ability of CD4 T cells to produce interferon-γ, tumor necrosis factor-α, and interleukin-2, their functional status, and their phenotype. The TB therapy determined a contraction of the triple functional CD4 T cells of the TBI subjects and active-TB patients. The CD45RA- CD27+ T cells stood out as a main subset of the Mtb-specific response in all groups. Before the TB-preventive therapy, the TBI subjects had higher proportion of Mtb-specific CD45RA-CD27+CD4+ T cells and the active-TB subjects had higher proportion of Mtb-specific CD45RA-CD27-CD4+ T cells compared to other groups. The TBI-IMID patients showed a phenotype similar to TBI, suggesting that the type of IMID and the IMID therapy did not affect the activation status of Mtb-specific CD4 T cells. Future studies on a larger and better-stratified TBI-IMID population will help to understand the change of the Mtb-specific immune response over time and to identify possible immune biomarkers of Mtb-containment or active replication.

Effect of HIV-infection on QuantiFERON-plus accuracy in patients with active tuberculosis and latent infection.

OBJECTIVE: HIV-infection increases the risk to progress to active-tuberculosis (TB). Detection of latent TB infection (LTBI) is needed to eventually propose preventive-therapy and reduce TB reservoir. QuantiFERON-TB Plus (QFT-Plus)-test identifies LTBI. Currently, only two studies on QFT-Plus accuracy in HIV-infected-population are available in high TB-endemic-countries. Therefore we aimed to evaluate the effect of HIV-infection on QFT-Plus accuracy to detect LTBI in a low TB-endemic-country. METHODS: We enrolled 465 participants, among the 167 HIV-infected-persons: 32 with active-TB (HIV-TB), 45 remote-LTBI (HIV-LTBI) and 90 at low M. tuberculosis (Mtb)-infection risk. Among the 298 HIV-uninfected-persons: 170 with active-TB, 76 recent-LTBI, 34 remote-LTBI and 18 with low Mtb-infection risk. RESULTS: QFT-Plus sensitivity was similar in TB regardless of HIV-status. CD4-count did not influence the distribution of IFN-γ values in HIV-TB and HIV-LTBI. Moreover HIV-LTBI and HIV-uninfected remote LTBI had a similar proportion of results in the uncertain range (IFNγ ≥0.2 ≤ 0.7 IU/ml) differently from those LTBI-persons reporting recent-exposure (p = 0.016). Cytometry results demonstrated that CD8-response was similar in HIV-infected- and -uninfected-persons whereas CD4-response was impaired in HIV-infected-persons (p = 0.011). CONCLUSIONS: HIV-infection does not affect QFT-Plus response in active-TB, whereas the time of exposure influences the proportion of uncertain-results in LTBI.

Characterization of QuantiFERON-TB-Plus results in latent tuberculosis infected patients with or without immune-mediated inflammatory diseases.

OBJECTIVES: Screening for latent tuberculosis infection (LTBI) diagnosis is mandatory in patients with immune-mediated inflammatory diseases (IMID) requiring biologics. QuantiFERON-TB-Plus (QFT-P), an LTBI diagnostic test, measures IFN-γ after M. tuberculosis-stimulation in TB1 and TB2 tubes in which a "CD4" or a "CD4 and CD8" response is respectively elicited. Aim of this study is to compare the response to QFT-P of IMID-LTBI patients candidates to a new biological therapy vs LTBI-subjects without IMID. METHODS: We prospectively enrolled 167 subjects: 61 IMID-LTBI and 106 NON-IMID-LTBI. RESULTS: All subjects were mitogen-responders. IFN-γ production was significantly lower in IMID-LTBI-patients compared to NON-IMID-LTBI-subjects. We observed discordant TB1 and TB2 results in 6.5% of IMID-LTBI-patients and in 8% of NON-IMID-LTBI-subjects. Applying a logistic regression analysis, we found that IMID-LTBI patients had a higher probability (TB1 stimulation OR 3.32; TB2 stimulation OR 4.33) to have IFNγ results ≤0.7 IU/mL compared to NON-IMID-LTBI-subjects. Interestingly, IMID-treatment did not interfere with the distribution of IFNγ-values. CONCLUSIONS: These results indicate that IMID-LTBI-patients have a low IFN-γ response to QFT-P, a high proportion of results ranging in the grey zone and a distribution of IFNγ-values independent from the IMID-treatment. These results are important for the management of LTBI screening in IMID patients.

Effect of therapy on Quantiferon-Plus response in patients with active and latent tuberculosis infection.

Lack of biomarkers for treatment monitoring is listed among the main requirements for next generation assays, as identified globally among tuberculosis (TB) researchers. In this study, we evaluated in a low TB endemic country such as Italy, the effect of preventive therapy on the results obtained in the QuantiFERON TB Plus (QFT-Plus), in a cohort of subjects with latent TB infection (LTBI) and active TB. We found that TB therapy significantly decreased IFN-γ values and number of responders to TB1- and TB2- peptides stimulation in both LTBI and active TB patients. Stratifying LTBI subjects according to the type of preventive TB therapy used, we found that INH treatment but not INH and RIF significantly decreased IFN-γ production. Stratifying the active TB patients according the microbiological status, we found that TB therapy significantly decreased IFN-γ response to antigen present in QFT-Plus test in patients with clinical diagnosis compared to those with a microbiological diagnosis. In conclusions, we demonstrated that TB therapy decreases IFN-γ level in response to antigen present in QFT-Plus test in LTBI and active TB patients. Future studies are needed to better characterize Mtb-specifc response as a potential marker for monitoring TB therapy and preventive treatment effects.

Combined use of Quantiferon and HBHA-based IGRA supports tuberculosis diagnosis and therapy management in children.

OBJECTIVES: Interferon-γ release assays (IGRA) are designed for diagnosis of tuberculosis (TB) infection, and do not discriminate latent TB infection (LTBI) from active TB. Heparin-binding hemagglutinin antigen (HBHA) emerged as a promising antigen for TB diagnosis when used in IGRA format. Aim of this study was to prospectively evaluate the performance of an HBHA-based IGRA to support TB diagnosis and TB therapy monitoring in children with TB infection or active TB disease. METHODS: Following clinical, microbiological and radiological assessment, children (0-14 years old) were tested by the QuantiFERON TB-Gold In tube (QFT) assay and an aliquot of whole-blood was stimulated with HBHA and IFNγ evaluated only in QFT-positive subjects. RESULTS: Among the 550 children tested, 486 (88.4%) scored negative and 64 (11.6%) positive. None of the QFT-negative had active TB. Among the QFT-positive, 45 were with LTBI and 19 active TB. HBHA-IGRA scored positive in 41/45 children (91.1%) with LTBI and in 6/19 active TB children (31.6%) at diagnosis (p = 0.001); remarkably, 5 of these 6 children with active TB scoring HBHA-positive were asymptomatic. Moreover, following TB-specific therapy, most of the non-HBHA-responding children, gained an HBHA-positive response. CONCLUSIONS: HBHA-based IGRA is a useful support in TB diagnosis and TB-therapy monitoring in children.

Interplay of DDP4 and IP-10 as a Potential Mechanism for Cell Recruitment to Tuberculosis Lesions.

INTRODUCTION: Mycobacterium tuberculosis is one of the world's most successful pathogens equipped to establish itself within the human host as a subclinical infection without overt disease. Unable to eradicate the bacteria, the immune system contains the infection in a granuloma structure. Th1 cells that are essential for infection control are recruited to the site of infection directed by chemokines, predominantly CXCL10. It has previously been shown that CXCL10 in the plasma of patients chronically infected with hepatitis C virus is present primarily in an antagonist form. This is due to N-terminal truncation by the enzyme DPP4, which results in the antagonist form that is capable of binding its receptor CXCR3, but does not induce signaling. We aimed to explore whether such CXCL10 antagonism may have an impact on the pathogenesis of tuberculosis (TB). RESULTS: We measured plasma levels of agonist and antagonist CXCL10 by Simoa digital ELISA, as well as DPP4 enzyme activity in the plasma of 20 patients with active TB infection, 10 patients with pneumonia infection, and a group of 10 healthy controls. We found higher levels of total and antagonist CXCL10 and reduced DPP4 enzyme activity in the plasma of TB patients compared to controls. We traced the source of CXCL10 secretion using immunohistochemical and confocal analysis to multinucleated giant cells in the TB lesions, and variable expression by macrophages. Interestingly, these cells were associated with DPP4-positive T cells. Moreover, the analysis of lymphocytes at the site of TB infection (bronchoalveolar lavage) showed a reduced frequency of CXCR3+ T cells. INTERPRETATION: Our data suggests that CXCL10 antagonism may be an important regulatory mechanism occurring at the site of TB pathology. CXCL10 can be inactivated shortly after secretion by membrane bound DPP4 (CD26), therefore, reducing its chemotactic potential. Given the importance of Th1 cell functions and IFN-γ-mediated effects in TB, our data suggest a possible unappreciated regulatory role of DPP4 in TB. PERSPECTIVES: DPP4 is the target for a class of enzyme inhibitors used in the treatment of diabetes, and the results from this study suggest that these drugs could be repurposed as an adjunct immunotherapy of patients with TB and MDR-TB.

Evaluation of IP-10 in Quantiferon-Plus as biomarker for the diagnosis of latent tuberculosis infection.

The QuantiFERON-TB Gold Plus (QFT-Plus) is a new test for latent tuberculosis infection (LTBI) diagnosis, in which has been added a new tube containing shorter peptides stimulating CD8 T-cells and CD4-stimulating-peptides. Measurement of alternative biomarkers to Interferon-γ (IFN-γ) in QFT-Plus may improve its sensitivity. Interferon-γ inducible protein 10 (IP-10), has been proposed as a tuberculosis (TB) biomarker. We aimed to evaluate the IP-10 accuracy in QFT-Plus for LTBI diagnosis. QFT-Plus was performed in 36 active TB, 31 LTBI and 16 healthy donors (HD). IP-10 was detected by ELISA. IP-10 is increased in TB1 and TB2 tubes in subjects with active TB and LTBI compared to HD. A ROC analysis comparing active TB and HD was performed and a cut-off of 1174 pg/mL for TB1 and 928.8 pg/mL for TB2 identified active TB with 86% sensitivity (Se) and 94% specificity (Sp). Moreover, increased IP-10 in response to TB1 was found in subjects with LTBI compared to those with active TB. A cut-off point of ≥16,108 pg/mL was chosen to maximize the test performance. However, the test predicted LTBI only with 58% Se and 61% Sp. These results suggest that IP-10 is an alternative biomarker to IFN-γ in the QFT-Plus format.

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

BACKGROUND: Human 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. AIM: to 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. METHODS: ART-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. RESULTS: The 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. CONCLUSIONS: After 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.

Impaired IFN-α-mediated signal in dendritic cells differentiates active from latent tuberculosis.

Individuals exposed to Mycobacterium tuberculosis (Mtb) may be infected and remain for the entire life in this condition defined as latent tuberculosis infection (LTBI) or develop active tuberculosis (TB). Among the multiple factors governing the outcome of the infection, dendritic cells (DCs) play a major role in dictating antibacterial immunity. However, current knowledge on the role of the diverse components of human DCs in shaping specific T-cell response during Mtb infection is limited. In this study, we performed a comparative evaluation of peripheral blood circulating DC subsets as well as of monocyte-derived Interferon-α DCs (IFN-DCs) from patients with active TB, subjects with LTBI and healthy donors (HD). The proportion of circulating myeloid BDCA3+ DCs (mDC2) and plasmacytoid CD123+ DCs (pDCs) declined significantly in active TB patients compared to HD, whereas the same subsets displayed a remarkable activation in LTBI subjects. Simultaneously, the differentiation of IFN-DCs from active TB patients resulted profoundly impaired compared to those from LTBI and HD individuals. Importantly, the altered developmental trait of IFN-DCs from active TB patients was associated with down-modulation of IFN-linked genes, marked changes in molecular signaling conveying antigen (Ag) presentation and full inability to induce Ag-specific T cell response. Thus, these data reveal an important role of IFN-α in determining the induction of Mtb-specific immunity.

Immune characterization of the HBHA-specific response in Mycobacterium tuberculosis-infected patients with or without HIV infection.

INTRODUCTION: RD1-based Interferon-γ Release Assays (IGRAs) cannot distinguish latent from active tuberculosis (TB) disease. Conversely, a positive response to heparin-binding haemagglutinin (HBHA)-based IGRAs, among TB-infected subjects, correlates with Mycobacterium tuberculosis (Mtb) containment and low risk of TB progression. The aim of this study was to characterize HBHA-immune responses in HIV-infected and uninfected subjects with active TB or latent TB infection (LTBI). METHODS: 49 subjects were prospectively enrolled: 22 HIV-uninfected (13 TB, 9 LTBI) and 27 HIV-infected (12 HIV-TB, 15 HIV-LTBI). Whole blood and peripheral blood mononuclear cells were stimulated with HBHA and RD1 antigens. Interferon (IFN)γ release was evaluated by ELISA whereas cytokine profile [IFNγ, tumor necrosis (TNF)α, interleukin (IL)2] and phenotype (CD45RA, CCR7) by flow cytometry. RESULTS: Among LTBI individuals, HBHA stimulation induced IFNγ release in all the HIV-uninfected, while, only 4/15 HIV-infected responded. Within the active TB, only 5/13 HIV-uninfected and 1/12 HIV-TB patients responded. Interestingly, by cytometry we showed that CD4+ T-cells response to HBHA was significantly impaired in the HIV-infected subjects with TB or LTBI compared to the HIV-uninfected subjects. The phenotype of HBHA-specific CD4 T-cells showed a predominantly central memory (CM) and effector memory (EM) phenotype without differences among the groups. Differently, HBHA-specific CD8+ T-cells, showed mainly a CM and naïve phenotype in LTBI group while TB, HIV-LTBI and HIV-TB groups were characterized by EM or terminally differentiated phenotypes. Interestingly, differently than what observed for RD1, the cytokine profile of HBHA-specific T-cells evaluated by cytometry showed that the CD4+ T-cells were mostly monofunctional. Conversely, CD8-specific T-cells were mostly monofunctional for both HBHA and RD1 stimulations. CONCLUSIONS: These results characterize the impact of HIV infection in CD4- and CD8-specific response to HBHA in both LTBI and TB patients. HIV infection impairs the CD4 response to HBHA and likely this may lead to an impairment of TB control.

First characterization of the CD4 and CD8 T-cell responses to QuantiFERON-TB Plus.

INTRODUCTION: QuantiFERON®-TB Gold Plus (QFT-Plus) is the new generation of QuantiFERON-TB Gold In-Tube test to identify latent tuberculosis infection (LTBI). QFT-Plus includes TB1 and TB2 tubes which contain selected Mycobacterium tuberculosis (Mtb) peptides designed to stimulate both CD4 and CD8 T-cells. Aim of this study is the flow cytometric characterization of the specific CD4 and CD8 T-cell responses to Mtb antigens contained within QFT-Plus. METHODS: We enrolled 27 active tuberculosis (TB) patients and 30 LTBI individuals. Following stimulation with TB1 and TB2, antigen-specific T-cells were characterized by flow cytometry. Data were also correlated with the grade of TB severity. RESULTS: TB1 mainly elicited a CD4 T-cell response while TB2 induced both CD4 and CD8 responses. Moreover, the TB2-specific CD4 response was detected for both active TB and LTBI patients, whereas the TB2-specific CD8 response was primarily associated with active TB (p = 0.01). CONCLUSIONS: To our knowledge, we report the first characterization of the CD4 and CD8 T-cell response to QFT-Plus. CD8 T-cell response is mainly due to TB2 stimulation which is largely associated to active TB. These results provide a better knowledge on the use of this assay.

Modulation of CD4 and CD8 response to QuantiFERON-TB Plus in patients with active tuberculosis and latent tuberculosis infection followed over time during treatment.

OBJECTIVE/BACKGROUND: Interferon (IFN)-γ release assays (IGRA) are designed for diagnosing tuberculosis (TB) infection. The new IGRA, QuantiFERON-TB Plus (QFT-Plus), is based on the enzyme-linked immunosorbent assay detection of IFN-γ after stimulation with Mycobacterium tuberculosis TB1 and TB2 antigens. TB1 elicits a cellular-mediated immune (CMI) response by CD4 T cells, and TB2 contains peptides recognized by both CD4 and CD8 T cells. The aim of the study is to characterize the CMI to QFT-Plus peptides in active TB and latent TB infection (LTBI) at baseline and during or after specific treatment (follow-up). METHODS: We enrolled 7 individuals with active TB and 11 individuals with LTBI at baseline and followed them during the treatment, either for active diseases or preventive therapy. Peripheral blood mononuclear cells were stimulated with QFT-Plus antigens (TB1, TB2, and mitogen). Cytokine profile (IFN-γ, tumor necrosis factor-α, interleukin-2) and phenotype (CD45RA, CD27) of CD4 and CD8 T cells were characterized by flow cytometry. RESULTS: All the individuals responded to mitogen. CD4 T-cell responses to TB1 and TB2 were similar in both individuals with active TB and those with LTBI evaluated over time. Differently, we found a higher number of TB2-associated CD8 T-cell responders in individuals with active TB than in those with LTBI. For individuals with active TB, there was no change in the specific response overtime. Differently, in individuals with LTBI, the number of CD8 responders to QFT-Plus antigens increased during preventive treatment (TB1=5/11 [45%], TB2=5/11 [45%]) compared with that at the time of enrolment (TB1=1/11 [9%], TB2=1/11 [9%]). Moreover, we analyzed the effector memory profile of T cells responding to QFT-Plus antigens. The largest component of antigen-specific CD4 T cells (65%) had a central memory (CD45RA-CD27+) phenotype at enrolment and during follow-up. In contrast, specific CD8 T cells, which were analyzed only at follow-up because they were almost absent at baseline, were characterized by a large component with naïve (CD45RA+CD27+) phenotype (40%) and a minor component with central memory (25%) features. CONCLUSION: To our knowledge, this is the first report characterizing CD4 and CD8 T-cell responses of individuals with active TB and with LTBI, followed overtime, to QFT-Plus antigens by flow cytometry. The results, although preliminary, may help in identifying better tools for monitoring therapy, especially in those with LTBI undergoing preventive treatment.

Modulation of interferon-gamma response to QuantiFERON-TB-plus detected by enzyme-linked immunosorbent assay in patients with active and latent tuberculosis infection.

OBJECTIVE/BACKGROUND: Interferon (IFN)-γ-release assays (IGRAs) are designed for the diagnosis of tuberculosis (TB) infection. The new IGRA called QuantiFERON-TB Plus (QFT-Plus) is based on enzyme-linked immunosorbent assay (ELISA) detection of IFN-γ following Myobacterium tuberculosis-antigen stimulation with TB1 and TB2 antigens. TB1 elicits a cell-mediated immune response by CD4 T cells and TB2 elicits a response from both CD4 and CD8 T cells. Here, we characterized variations IFN-γ release detected by ELISA to QFT-IT and QFT-Plus in patients with active TB and latent TB infection (LTBI) at baseline and during or after specific treatment (follow-up). METHODS: We studied seven patients with active TB and 10 patients with LTBI at baseline and during treatment either for active disease or preventive therapy. IFN-γ release detected by ELISA to QFT-IT and QFT-Plus was concomitantly evaluated over time. Statistical analysis was performed using a nonparametrical test for a paired dataset (Wilcoxon test). RESULTS: All participants responded to the mitogen, with all active-TB patients responding to QFT-IT or QFT-Plus at baseline. The responses did not change over time either qualitatively (number of responders) or quantitatively (IFN-γ release evaluated as IU/mL). Among the LTBI group, although all participants responded to both QFT-IT and QFT-Plus and the responses did not change over time, the quantitative responses to QFT-Plus showed a different trend. Specifically, response to TB2 was significantly lower at follow-up as compared with that observed at baseline (p=0.004), whereas the response to TB1 was not significantly different (p=0.16). CONCLUSION: To our knowledge, this is the first report characterizing IFN-γ responses to QFT-Plus antigens in participants with active TB and LTBI over time. The data need to be confirmed in larger settings; however, we showed that monitoring IFN-γ release in response to TB2 can be used to evaluate preventive therapy immune changes. This can be useful also as a tool for public health control strategies in settings where preventive treatment is recommended.

Characterization of the CD4 and CD8 T-cell response in the QuantiFERON-TB Gold Plus kit.

OBJECTIVE/BACKGROUND: QuantiFERON-TB Gold In-Tube (QFT-GIT, Qiagen, Hilden, Germany) is an interferon-γ (IFN-γ) release assay designed to detect latent tuberculosis infection (LTBI). Although QFT-GIT has several advantages (mainly that it is not affected by the Bacille Calmette-Guérin vaccination), it has a poor sensitivity in immune-compromised individuals as it involves an immune response-based detection. Recently, QuantiFERON-TB Gold Plus (QFT-Plus) assay has been proposed as a new generation of QFT-GIT. QFT-Plus includes two tubes, TB1 and TB2 with Mycobacterium tuberculosis antigens to elicit a specific immune response. TB1 contains peptides derived from the antigens 6kDa early secretory antigenic target (ESAT-6) and 10kDa culture filtrate protein (CFP-10) (TB-7.7, present in QFT-GIT, has been removed), and it is designed to induce a specific CD4 T-cell response. TB2 contains newly designed peptides stimulating IFN-γ production by both CD4 and CD8 T cells. The additional peptides for eliciting CD8 T-cell responses have been included to increase the sensitivity of the test for LTBI detection. The aim of the study was to evaluate specific CD4 and CD8 T-cell responses to the M. tuberculosis antigens contained within the QFT-Plus test by flow cytometry in individuals with active TB and LTBI. METHODS: We enrolled 23 individuals with active TB and 30 individuals with LTBI. QFT-Plus assay and intracellular staining were performed. One million of peripheral blood mononuclear cells in 1ml of complete medium (RPMI 1640) were dispensed in QFT-Plus tubes. Following 16-24h stimulation, antigen-specific T cells were characterized by flow cytometry evaluating CD4, CD8, CD3 markers, and IFN-γ production. For statistical analysis, nonparametric tests were performed. RESULTS: We found that CD4 T-cell responses were induced by both TB1 and TB2. Differently, the CD8 T-cell response was mainly induced by TB2 and was significantly higher than that induced by TB1 (p=0.01). The frequency of Mtb specific T-cells observed in individuals with active TB was significantly higher than in those with LTBI (p=0.04). Finally, TB2-specific CD8 T-cell responses in individuals with active TB were associated with high radiological severity of lung lesions and microbiological diagnosis (based on M. tuberculosis isolation in sputum culture). CONCLUSION: This is the first characterization of CD4 and CD8 T-cell responses to QFT-Plus TB1 and TB2 tubes in individuals with active TB and LTBI enrolled in a low TB-endemic country such as Italy. We demonstrated that the increased sensitivity is a consequence of the ability of TB2 to induce a CD8 T-cell response which is mainly associated with active TB. This assay has the potential to be very useful in conditions of immune depression due to CD4 T-cell impairments.

Polyfunctional T-cells and effector memory phenotype are associated with active TB in HIV-infected patients.

OBJECTIVES: Polyfunctional T-cells associate with chronic viral infection control while their involvement in tuberculosis (TB) is unclear. We evaluated TB-specific polyfunctional T-cell response and memory status in antiretroviral treatment (ART)-naïve HIV-infected patients from a low TB-endemic country. METHODS: We prospectively enrolled HIV-infected patients, 12 with active TB (HIV-TB) and 15 with latent tuberculosis infection (LTBI). Peripheral blood cells were stimulated with TB antigens (RD1 proteins/peptides), HIV antigens, cytomegalovirus (CMV) and staphylococcal enterotoxin B (SEB) and analyzed by cytometry. RESULTS: The HIV-TB showed a higher frequency of polyfunctional CD4(+) T-cells in response to RD1 antigens than HIV-LTBI (p = 0.007). Among the CD8(+) T-cells, both groups showed a significantly higher frequency of RD1-specific monofunctional cells than polyfunctional cells (p = 0.03). Analyzing the cytokine profile, IFNγ(+) TNFα(+) CD4(+) T-cells associated with HIV-TB (p ≤ 0.02) whereas IL2(+) TNFα(+) associated with HIV-LTBI (p = 0.009). CD4(+) T-cell response presented an effector-memory status in HIV-TB (p = 0.007) and an effector-memory terminally-differentiated phenotype in HIV-LTBI (p = 0.03). CD8(+) T-cell response presented an effector status in HIV-LTBI (p = 0.02). No significant cytokine profile pattern associated with responses to the other stimuli tested. CONCLUSIONS: In HIV-infection, polyfunctional CD4(+) T-cell-response associates with active TB, characterized by a high proportion of IFNγ(+) TNFα(+) and an effector-memory phenotype.

High urine IP-10 levels associate with chronic HCV infection.

OBJECTIVES: Independent of IL-28B polymorphisms, blood IP-10 is a promising biomarker for predicting therapy response in chronic HCV infection. Urine IP-10 has been proposed as a biomarker in tuberculosis, but to date, no urine biomarkers for HCV infection have been evaluated. In this cross-sectional study, we assessed whether IP-10 is detectable in the urine of chronically HCV-infected patients, and if so, whether urine IP-10 correlates with serum IP-10 and HCV-specific clinical parameters. METHODS: IP-10 was measured by ELISA in serum and urine concomitantly taken from 38 HCV-viremic patients, 10 cured-HCV subjects and 11 healthy donors enrolled as controls. RESULTS: The urine of HCV-viremic patients showed measurable amounts of IP-10, although significantly lower than in serum (p < 0.0001). Urine IP-10 was normalized with creatinuria levels and we found that the urine IP-10/creatinuria ratio was significantly higher in HCV-viremic patients than in cured-HCV subjects (p = 0.002) and healthy donors (p = 0.008), and that it significantly correlated with transaminases (p = 0.01), although the correlation was low. Similarly, the serum IP-10 level significantly associated with HCV-viremic patients (p < 0.0001) and correlated with transaminases (p < 0.0001). CONCLUSIONS: For the first time to our knowledge, we show that IP-10 is detected and increased in the urine of HCV-viremic patients compared to healthy donors and cured-HCV subjects.

Higher frequency of T-cell response to M. tuberculosis latency antigen Rv2628 at the site of active tuberculosis disease than in peripheral blood.

RATIONALE: Due to the invasive nature of the procedures involved, most studies of Mycobacterium tuberculosis (Mtb)-specific immunity in humans have focused on the periphery rather than the site of active infection, the lung. Recently, antigens associated with Mtb-latency and -dormancy have been described using peripheral blood (PB) cells; however their response in the lung is unknown. The objective of this report was to evaluate, in patients prospectively enrolled with suspected active tuberculosis (TB), whether the latency antigen Rv2628 induces local-specific immune response in bronchoalveolar lavage (BAL) cells compared to PB cells. MATERIAL/METHODS: Among the 41 subjects enrolled, 20 resulted with active TB. Among the 21 without active disease, 9 were defined as subjects with latent TB-infection (LTBI) [Quantiferon TB Gold In-tube positive]. Cytokine responses to Rv2628 were evaluated by enzyme linked immunospot (ELISPOT) assay and flow cytometric (FACS) analysis. RD1-secreted antigen stimulation was used as control. RESULTS: There was a significantly higher frequency of Rv2628- and RD1-specific CD4+ T-cells in the BAL of active TB patients than in PB. However the trend of the response to Rv2628 in subjects with LTBI was higher than in active TB in both PB and BAL, although this difference was not significant. In active TB, Rv2628 and RD1 induced a cytokine-response profile mainly consisting of interferon (IFN)-γ-single-positive over double-IFN-γ/interleukin (IL)-2 T-cells in both PB and BAL. Finally, BAL-specific CD4+ T-cells were mostly effector memory (EM), while peripheral T-cell phenotypes were distributed among naïve, central memory and terminally differentiated effector memory T-cells. CONCLUSIONS: In this observational study, we show that there is a high frequency of specific T-cells for Mtb-latency and RD1-secreted antigens (mostly IFN-γ-single-positive specific T-cells with an EM phenotype) in the BAL of active TB patients. These data may be important for better understanding the pathogenesis of TB in the lung.

Methylated HBHA produced in M. smegmatis discriminates between active and non-active tuberculosis disease among RD1-responders.

BACKGROUND: A challenge in tuberculosis (TB) research is to develop a new immunological test that can help distinguish, among subjects responsive to QuantiFERON TB Gold In tube (QFT-IT), those who are able to control Mtb replication (remote LTBI, recent infection and past TB) from those who cannot (active TB disease). IFN-γ response to the Heparin-binding-hemagglutinin (HBHA) of Mtb has been associated with LTBI, but the cumbersome procedures of purifying the methylated and immunological active form of the protein from Mtb or M. bovis Bacillus Calmette et Guerin (BCG) have prevented its implementation in a diagnostic test. Therefore, the aim of the present study was to evaluate the IFN-γ response to methylated HBHA of Mtb produced in M. smegmatis (rHBHAms) in individuals at different stages of TB who scored positive to QFT-IT. METHODOLOGY/PRINCIPAL FINDINGS: 87 individuals at different stages of TB who scored positive to QFT-IT were selected. IFN-γ response to in vitro whole blood stimulation with rHBHAms was evaluated by short-term and long-term tests and detected by ELISA or flow cytometry. We demonstrated that the IFN-γ response to rHBHAms is mediated by CD4(+) T-cells with an effector-memory phenotype. This response, evaluated by short-term-tests, is significantly lower in active TB than in remote LTBI (p = 0.0010) and past TB (p = 0.0152). These results were confirmed by long-term tests. The qualitative data confirmed that IFN-γ responses higher than the cut-off point identified by ROC analysis are associated with the status of non-active disease. CONCLUSIONS: In this study we show that the T-cell response to a recombinant and methylated HBHA of Mtb produced in M. smegmatis is useful to discriminate between active and non-active TB disease among those responsive to QFT-IT in a whole blood system. Further studies are needed to improve the accuracy of the assay.

IP-10 detection in urine is associated with lung diseases.

BACKGROUND: Blood cytokines and chemokines have been proposed as biomarkers for tuberculosis (TB). Recently, some immune mediators found in the urine of patients with renal dysfunctions have also been suggested as potential biomarkers. Finding biomarkers for TB in urine would present several advantages over blood in terms of collection and safety. The objective of this study was to investigate the presence of cytokines and chemokines in the urine of patients with pulmonary TB at the time of diagnosis. In a subgroup, the evaluation was also performed during TB treatment and at therapy completion. Patients with lung diseases other than TB, and healthy subjects were also enrolled. METHODS: Urine samples from 138 individuals, after exclusion of renal dysfunctions, were collected during an 18 month-period. Among them, 58 received a diagnosis of pulmonary TB, 28 resulted having lung diseases other than TB, and 34 were healthy subjects. Moreover, 18 TB patients, 9 of whom were tested 2 months after AFB smear sputum reversion and 9 of whom were cured of TB were also included. Cytokines and chemokines in urine were evaluated using a Cytometric-Bead-Array-Flex-Set. IP-10 detection in 49 subjects was also carried out in parallel by using an Enzyme Linked ImmunoSorbent Assay (ELISA). RESULTS: IFN-γ, TNF-α, IL-2, IL-8, MIP-1α, MIP-1ß and RANTES were poorly detected in all urine samples. Conversely, IP-10 was consistently detected in urine and its level was significantly increased in patients with lung disease compared to healthy subjects (p < 0.001). Increased IP-10 levels were found in both pulmonary TB and lung diseases other than TB. Moreover lower IP-10 levels were found in cured-TB patients compared to the levels at the time of diagnosis, and this difference was close to significance (p = 0.06). Interestingly, we demonstrated a significant correlation between the data obtained by flow cytometry and ELISA (r² 0.82, p < 0.0001). CONCLUSIONS: IP-10, in contrast to IFN-γ, TNF-α, IL-2, IL-8, MIP-1α, MIP-1ß and RANTES, is detectable in the urine of patients with pulmonary diseases in the absence of renal dysfunctions. Moreover, the IP-10 level in cured-TB patients is comparable to that found in healthy subjects. More studies are needed to further investigate the clinical utility of these findings.

New tools for detecting latent tuberculosis infection: evaluation of RD1-specific long-term response.

BACKGROUND: Interferon-gamma (IFN-gamma) release assays (IGRAs) were designed to detect latent tuberculosis infection (LTBI). However, discrepancies were found between the tuberculin skin test (TST) and IGRAs results that cannot be attributed to prior Bacille Calmètte Guerin vaccinations. The aim of this study was to evaluate tools for improving LTBI diagnosis by analyzing the IFN-gamma response to RD1 proteins in prolonged (long-term response) whole blood tests in those subjects resulting negative to assays such as QuantiFERON-TB Gold In tube (QFT-IT). METHODS: The study population included 106 healthy TST+ individuals with suspected LTBI (recent contact of smear-positive TB and homeless) consecutively enrolled. As controls, 13 healthy subjects unexposed to M. tuberculosis (TST-, QFT-IT-) and 29 subjects with cured pulmonary TB were enrolled. IFN-gamma whole blood response to RD1 proteins and QFT-IT were evaluated at day 1 post-culture. A prolonged test evaluating long-term IFN-gamma response (7-day) to RD1 proteins in diluted whole blood was performed. RESULTS: Among the enrolled TST+ subjects with suspected LTBI, 70/106 (66.0%) responded to QFT-IT and 64/106 (60.3%) to RD1 proteins at day 1. To evaluate whether a prolonged test could improve the detection of LTBI, we set up the test using cured TB patients (with a microbiologically diagnosed past pulmonary disease) who resulted QFT-IT-negative and healthy controls as comparator groups. Using this assay, a statistically significant difference was found between IFN-gamma levels in cured TB patients compared to healthy controls (p < 0.006). Based on these data, we constructed a receiver operating characteristic (ROC) curve and we calculated a cut-off. Based on the cut-off value, we found that among the 36 enrolled TST+ subjects with suspected LTBI not responding to QFT-IT, a long term response to RD1 proteins was detected in 11 subjects (30.6%). CONCLUSION: These results indicate that IFN-gamma long-term response to M. tuberculosis RD1 antigens may be used to detect past infection with M. tuberculosis and may help to identify additional individuals with LTBI who resulted negative in the short-term tests. These data may provide useful information for improving immunodiagnostic tests for tuberculosis infection, especially in individuals at high risk for active TB.