Immune-based assays are promising tools to help to formulate diagnosis of active tuberculosis. A multiparameter flow cytometry assay assessing T-cell responses specific to Mycobacterium tuberculosis and the combination of both CD4 and CD8 T-cell responses accurately discriminated between active tuberculosis and latent infection.

BACKGROUND. The identification and treatment of individuals with tuberculosis (TB) is a global public health priority. Accurate diagnosis of pulmonary active TB (ATB) disease remains challenging and relies on extensive medical evaluation and detection of Mycobacterium tuberculosis (Mtb) in the patient's sputum. Further, the response to treatment is monitored by sputum culture conversion, which takes several weeks for results. Here, we sought to identify blood-based host biomarkers associated with ATB and hypothesized that immune activation markers on Mtb-specific CD4⁺ T cells would be associated with Mtb load in vivo and could thus provide a gauge of Mtb infection. METHODS. Using polychromatic flow cytometry, we evaluated the expression of immune activation markers on Mtb-specific CD4⁺ T cells from individuals with asymptomatic latent Mtb infection (LTBI) and ATB as well as from ATB patients undergoing anti-TB treatment. RESULTS. Frequencies of Mtb-specific IFN-γ⁺CD4⁺ T cells that expressed immune activation markers CD38 and HLA-DR as well as intracellular proliferation marker Ki-67 were substantially higher in subjects with ATB compared with those with LTBI. These markers accurately classified ATB and LTBI status, with cutoff values of 18%, 60%, and 5% for CD38⁺IFN-γ⁺, HLA-DR⁺IFN-γ⁺, and Ki-67⁺IFN-γ⁺, respectively, with 100% specificity and greater than 96% sensitivity. These markers also distinguished individuals with untreated ATB from those who had successfully completed anti-TB treatment and correlated with decreasing mycobacterial loads during treatment. CONCLUSION. We have identified host blood-based biomarkers on Mtb-specific CD4⁺ T cells that discriminate between ATB and LTBI and provide a set of tools for monitoring treatment response and cure. TRIAL REGISTRATION. Registration is not required for observational studies.

Background: Vaccination that prevents tuberculosis (TB) disease, particularly in adolescents, would have the greatest impact on the global TB epidemic. Safety, reactogenicity and immunogenicity of the vaccine candidate M72/AS01 E was evaluated in healthy, HIV-negative adolescents in a TB endemic region, regardless of Mycobacterium tuberculosis (M.tb) infection status. Methods: In a phase II, double-blind randomized, controlled study (NCT00950612), two doses of M72/AS01 E or placebo were administered intramuscularly, one month apart. Participants were followed-up post-vaccination, for 6 months. M72-specific immunogenicity was evaluated by intracellular cytokine staining analysis of T cells and NK cells by flow cytometry. Results: No serious adverse events were recorded. M72/AS01 E induced robust T cell and antibody responses, including antigen-dependent NK cell IFN-γ production. CD4 and CD8 T cell responses were sustained at 6 months post vaccination. Irrespective of M.tb infection status, vaccination induced a high frequency of M72-specific CD4 T cells expressing multiple combinations of Th1 cytokines, and low level IL-17. We observed rapid boosting of immune responses in M.tb-infected participants, suggesting natural infection acts as a prime to vaccination. Conclusions: The clinically acceptable safety and immunogenicity profile of M72/AS01 E in adolescents living in an area with high TB burden support the move to efficacy trials.

Heterologous prime-boost strategies hold promise for vaccination against tuberculosis. However, the T-cell characteristics required for protection are not known. We proposed that boost vaccines should induce long-lived functional and phenotypic changes to T cells primed by Bacille Calmette Guerin (BCG) and/or natural exposure to mycobacteria. We characterized changes among specific CD4 + T cells after vaccination with the MVA85A vaccine in adults, adolescents, and children. CD4 + T cells identified with Ag85A peptide-bearing HLA class II tetramers were characterized by flow cytometry. We also measured proliferative potential and cytokine expression of Ag85A-specific CD4 + T cells. During the effector phase, MVA85A-induced specific CD4 + T cells coexpressed IFN-γ and IL-2, skin homing integrins, and the activation marker CD38. This was followed by contraction and a transition to predominantly IL-2-expressing, CD45RA - CCR7 + CD27 + or CD45RA + CCR7 + CD27 + specific CD4 + T cells. These surface phenotypes were similar to Ag85A-specific T cells prior to MVA85A. However, functional differences were observed postvaccination: specific proliferative capacity was markedly higher after 6-12 months than before vaccination. Our data suggest that MVA85A vaccination may modulate Ag85A-specific CD4 + T-cell function, resulting in greater recall potential. Importantly, surface phenotypes commonly used as proxies for memory T-cell function did not associate with functional effects of vaccination. © 2013 The Authors.

CD8 T cells play a critical role in control of chronic viral infections; however, the role of these cells in containing persistent bacterial infections, such as those caused by Mycobacterium tuberculosis (Mtb), is less clear. We assessed the phenotype and functional capacity of CD8 T cells specific for the immunodominant Mtb antigens CFP-10 and ESAT-6, in patients with pulmonary tuberculosis (TB) disease, before and after treatment, and in healthy persons with latent Mtb infection (LTBI). In patients with TB disease, CFP-10/ESAT-6-specific IFN-γ+ CD8 T cells had an activated, pro-apoptotic phenotype, with lower Bcl-2 and CD127 expression, and higher Ki67, CD57, and CD95 expression, than in LTBI. When CFP-10/ESAT-6-specific IFN-γ+ CD8 T cells were detectable, expression of distinct combinations of these markers was highly sensitive and specific for differentiating TB disease from LTBI. Successful treatment of disease resulted in changes of these markers, but not in restoration of CFP-10/ESAT-6-specific CD8 or CD4 memory T cell proliferative capacity. These data suggest that high mycobacterial load in active TB disease is associated with activated, short-lived CFP-10/ESAT-6-specific CD8 T cells with impaired functional capacity that is not restored following treatment. By contrast, LTBI is associated with preservation of long-lived CFP-10/ESAT-6-specific memory CD8 T cells that maintain high Bcl-2 expression and which may readily proliferate.

The Notch ligand delta-like 4 (DLL4) is known to fine-tune the CD4+ T cell cytokine response. DLL4 is expressed on the surface of antigen-presenting cells (APCs) in a MyD88-dependent manner. We found that DLL4 expression was upregulated on bone marrow progenitor cells and APCs in mice infected with BCG Mycobacterium. Transfer of DLL4+ progenitor cells from infected hosts resulted in an increase DLL4+ myeloid cells in the spleen, indicating that expression of the dll4 gene is propagated throughout hematopoiesis. We also found an increase in DLL4+ monocytes from individuals who were infected with Mycobacterium tuberculosis. In latent individuals, DLL4 expression correlated with increased cytokine production from T cells in response to PPD stimulation. Finally, antibody blockade of DLL4 reduced T cell cytokine production from naïve T cells stimulated with antigen. These results demonstrate that the Notch ligand DLL4 can influence T cell cytokine production in both humans and mice, and further reveal that expression of DLL4 is upregulated on early hematopoietic progenitors in response to chronic mycobacterial infection. These data suggest that widespread DLL4 expression may occur as a result of mycobacterial infection, and that this expression may alter CD4+ T cell responses to both previously encountered and novel antigens.

Persistent antigen stimulation in chronic infections has been associated with antigen-specific T cell dysfunction and upregulation of inhibitory receptors, including programmed cell death protein 1 (PD-1). Pulmonary tuberculosis (TB) disease is characterized by high levels of Mycobacterium tuberculosis (Mtb), yet the relationship between bacterial load, PD-1 expression, and Mtb-specific T cell function in human TB has not been well-defined. Using peripheral blood samples from adults with LTBI and with pulmonary TB disease, we tested the hypothesis that PD-1 expression is associated with bacterial load and functional capacity of Mtb-specific T cell responses. We found that PD-1 was expressed at significantly higher levels on Th1 cytokine-producing Mtb-specific CD4 T cells from patients with smear-positive TB, compared with smear-negative TB and LTBI, which decreased after completion of anti-TB treatment. By contrast, expression of PD-1 on Mtb-specific CD8 T cells was significantly lower than on Mtb-specific CD4 T cells and did not differ by Mtb infection and disease status. In vitro stimulation of PBMC with Mtb antigens demonstrated that PD-1 is induced on proliferating Mtb-specific CD4 T cells and that Th1 cytokine production capacity is preferentially maintained within PD-1+ proliferating CD4 T cells, compared with proliferating Mtb-specific CD4 T cells that lack PD-1 expression. Together, these data indicate that expression of PD-1 on Mtb-specific CD4 T cells is indicative of mycobacterial antigen exposure and identifies a population of effector cells with Th1 cytokine production capacity. These studies provide novel insights into the role of the PD-1 pathway in regulating CD4 and CD8 T cell responses in Mtb infection and provide rationale for future studies to evaluate PD-1 expression on antigen-specific CD4 T cells as a potential biomarker for bacterial load and treatment response in human TB.

Chronic infections in mice can result in defects in memory CD8 T cell properties including low expression of the IL-7Rα (CD127). To determine whether defects in memory CD8 T cell formation exist during human chronic infections and to what extent these defects may be allele- or epitope-specific, we compared influenza (Flu), vaccinia (VV) and EBV-specific CD8 T cells to HIV-specific CD8 T cells, using a panel of 13 HIV tetramers. Compared to Flu, VV or EBV, HIV tetramer+ CD8 T cells expressed significantly lower levels of CD127, and this reduction was pervasive across all epitopes and alleles tested and over a wide range of viral loads and CD4 counts. These results indicate impaired HIV-specific memory CD8 T cell differentiation, regardless of level of control of viremia, epitopes targeted or restricting HLA alleles.

While a third of the world carries the burden of tuberculosis, disease control has been hindered by a lack of tools, including a rapid, point-of-care diagnostic and a protective vaccine. In many infectious diseases, antibodies (Abs) are powerful biomarkers and important immune mediators. However, in Mycobacterium tuberculosis (Mtb) infection, a discriminatory or protective role for humoral immunity remains unclear. Using an unbiased antibody profiling approach, we show that individuals with latent tuberculosis infection (Ltb) and active tuberculosis disease (Atb) have distinct Mtb-specific humoral responses, such that Ltb infection is associated with unique Ab Fc functional profiles, selective binding to FcγRIII, and distinct Ab glycosylation patterns. Moreover, compared to Abs from Atb, Abs from Ltb drove enhanced phagolysosomal maturation, inflammasome activation, and, most importantly, macrophage killing of intracellular Mtb. Combined, these data point to a potential role for Fc-mediated Ab effector functions, tuned via differential glycosylation, in Mtb control.