REL and BHLHE40 Variants Are Associated with IL-12 and IL-10 Responses and Tuberculosis Risk.

The major human genes regulating Mycobacterium tuberculosis-induced immune responses and tuberculosis (TB) susceptibility are poorly understood. Although IL-12 and IL-10 are critical for TB pathogenesis, the genetic factors that regulate their expression in humans are unknown. CNBP, REL, and BHLHE40 are master regulators of IL-12 and IL-10 signaling. We hypothesized that common variants in CNBP, REL, and BHLHE40 were associated with IL-12 and IL-10 production from dendritic cells, and that these variants also influence adaptive immune responses to bacillus Calmette-Guérin (BCG) vaccination and TB susceptibility. We characterized the association between common variants in CNBP, REL, and BHLHE40, innate immune responses in dendritic cells and monocyte-derived macrophages, BCG-specific T cell responses, and susceptibility to pediatric and adult TB in human populations. BHLHE40 single-nucleotide polymorphism (SNP) rs4496464 was associated with increased BHLHE40 expression in monocyte-derived macrophages and increased IL-10 from peripheral blood dendritic cells and monocyte-derived macrophages after LPS and TB whole-cell lysate stimulation. SNP BHLHE40 rs11130215, in linkage disequilibrium with rs4496464, was associated with increased BCG-specific IL-2+CD4+ T cell responses and decreased risk for pediatric TB in South Africa. SNPs REL rs842634 and rs842618 were associated with increased IL-12 production from dendritic cells, and SNP REL rs842618 was associated with increased risk for TB meningitis. In summary, we found that genetic variations in REL and BHLHE40 are associated with IL-12 and IL-10 cytokine responses and TB clinical outcomes. Common human genetic regulation of well-defined intermediate cellular traits provides insights into mechanisms of TB pathogenesis.

Qualification of the differential leukocyte count and immunophenotyping in cryopreserved ex vivo whole blood assay.

We developed a flow cytometry-based assay, termed Differential Leukocyte Counting and Immunophenotyping in Cryopreserved Ex vivo whole blood (DLC-ICE), that allows quantification of absolute counts and frequencies of leukocyte subsets and measures expression of activation, phenotypic and functional markers. We evaluated the performance of the DLC-ICE assay by determining inter-operator variability for processing fresh whole blood (WB) from healthy donors collected at multiple clinical sites. In addition, we assessed inter-operator variability for staining of fixed cells and robustness across different anticoagulants. Accuracy was evaluated by comparing DLC-ICE measurements to real-time cell enumeration using an accredited hematology analyzer. Finally, we developed and tested the performance of a 27-colour immunophenotyping panel on cryopreserved fixed WB and compared results to matched fresh WB. Overall, we observed <20% variability in absolute counts and frequencies of granulocytes, monocytes and lymphocytes (T, B and NK cells) when fresh WB was collected in different anti-coagulant tubes, processed or stained by independent operators. Absolute cell counts measured across operators and anti-coagulants using the DLC-ICE method exhibited excellent correlation with the reference method, complete blood count (CBC) with differential, measured using a hematology analyzer (r2 > 0.9 for majority of measurements). A comparison of leukocyte immunophenotyping on fresh WB versus DLC-ICE processed blood yielded equivalent and linear results over a wide dynamic range (r2 = 0.94 over 10-104 cells/µL). These results demonstrate low variability across trained operators, high robustness, linearity and accuracy, supporting utility of the DLC-ICE assay for large cohort studies involving multiple clinical research sites.

MR1-Independent Activation of Human Mucosal-Associated Invariant T Cells by Mycobacteria.

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).

Peripheral Blood Mucosal-Associated Invariant T Cells in Tuberculosis Patients and Healthy Mycobacterium tuberculosis-Exposed Controls.

BACKGROUND: In human blood, mucosal-associated invariant T (MAIT) cells are abundant T cells that recognize antigens presented on non-polymorphic major histocompatibility complex-related 1 (MR1) molecules. The MAIT cells are activated by mycobacteria, and prior human studies indicate that blood frequencies of MAIT cells, defined by cell surface markers, decline during tuberculosis (TB) disease, consistent with redistribution to the lungs. METHODS: We tested whether frequencies of blood MAIT cells were altered in patients with TB disease relative to healthy Mycobacterium tuberculosis-exposed controls from Peru and South Africa. We quantified their frequencies using MR1 tetramers loaded with 5-(2-oxopropylideneamino)-6-D-ribitylaminouracil. RESULTS: Unlike findings from prior studies, frequencies of blood MAIT cells were similar among patients with TB disease and latent and uninfected controls. In both cohorts, frequencies of MAIT cells defined by MR1-tetramer staining and coexpression of CD161 and the T-cell receptor alpha variable gene TRAV1-2 were strongly correlated. Disease severity captured by body mass index or TB disease transcriptional signatures did not correlate with MAIT cell frequencies in patients with TB. CONCLUSIONS: Major histocompatibility complex (MHC)-related 1-restrictied MAIT cells are detected at similar levels with tetramers or surface markers. Unlike MHC-restricted T cells, blood frequencies of MAIT cells are poor correlates of TB disease but may play a role in pathophysiology.

Mtb specific HLA-E restricted T cells are induced during Mtb infection but not after BCG administration in non-human primates and humans.

Novel vaccines targeting the world's deadliest pathogen Mycobacterium tuberculosis (Mtb) are urgently needed as the efficacy of the Bacillus Calmette-Guérin (BCG) vaccine in its current use is limited. HLA-E is a virtually monomorphic unconventional antigen presentation molecule and HLA-E restricted Mtb specific CD8+ T cells can control intracellular Mtb growth, making HLA-E a promising vaccine target for Mtb. In this study, we evaluated the frequency and phenotype of HLA-E restricted Mtb specific CD4+/CD8+ T cells in the circulation and bronchoalveolar lavage fluid of two independent non-human primate (NHP) studies and from humans receiving BCG either intradermally or mucosally. BCG vaccination followed by Mtb challenge in NHPs did not affect the frequency of circulating and local HLA-E/Mtb CD4+ and CD8+ T cells, and we saw the same in humans receiving BCG. HLA-E/Mtb T cell frequencies were significantly increased after Mtb challenge in unvaccinated NHPs, which was correlated with higher TB pathology. Together, HLA-E/Mtb restricted T cells are minimally induced by BCG in humans and rhesus macaques (RMs) but can be elicited after Mtb infection in unvaccinated RMs. These results give new insights into targeting HLA-E as a potential immune mechanism against TB.

High expression of midkine in the airways of patients with cystic fibrosis.

Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene result in impaired host defense during cystic fibrosis (CF), where Pseudomonas aeruginosa becomes a key pathogen. We investigated the expression pattern of the antibacterial growth factor midkine (MK) in CF and the possible interference with its activity by the altered airway microenvironment. High MK expression was found in CF lung tissue compared with control samples, involving epithelia of the large and small airways, alveoli, and cells of the submucosa (i.e., neutrophils and mast cells). In CF sputum, MK was present at 100-fold higher levels, but was also subject to increased degradation, compared with MK in sputum from healthy control subjects. MK exerted a bactericidal effect on P. aeruginosa, but increasing salt concentrations and low pH impaired this activity. Molecular modeling suggested that the effects of salt and pH were attributable to electrostatic screening and a charge-neutralization of the membrane, respectively. Both the neutrophil elastase and elastase of P. aeruginosa cleaved MK to smaller fragments, resulting in impaired bactericidal activity. Thus, MK is highly expressed in CF, but its bactericidal properties may be impaired by the altered microenvironment, as reflected by the in vitro conditions used in this study.

Effects of BCG vaccination on donor unrestricted T cells in two prospective cohort studies.

BACKGROUND: Non-protein antigen classes can be presented to T cells by near-monomorphic antigen-presenting molecules such as CD1, MR1, and butyrophilin 3A1. Such T cells, referred to as donor unrestricted T (DURT) cells, typically express stereotypic T cell receptors. The near-unrestricted nature of DURT cell antigen recognition is of particular interest for vaccine development, and we sought to define the roles of DURT cells, including MR1-restricted MAIT cells, CD1b-restricted glucose monomycolate (GMM)-specific T cells, CD1d-restricted NKT cells, and γδ T cells, in vaccination against Mycobacterium tuberculosis. METHODS: We compared and characterized DURT cells following primary bacille Calmette-Guerin (BCG) vaccination in a cohort of vaccinated and unvaccinated infants, as well as before and after BCG-revaccination in adults. FINDINGS: 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, primary BCG vaccination was associated with increased frequencies of γδ T cells as well as a novel subset of CD26+CD161+TRAV1-2- IFN-γ-expressing CD4+ T cells in infants. INTERPRETATION: Our findings, that most DURT cell populations were not modulated by BCG, do not preclude a role of BCG in modulating other qualitative aspects of DURT cells. More studies are required to understand the full potential of DURT cells in new TB vaccine strategies. FUNDING: Aeras, the National Institutes of Health, and the Bill and Melinda Gates Foundation.

Functional and Activation Profiles of Mucosal-Associated Invariant T Cells in Patients With Tuberculosis and HIV in a High Endemic Setting.

Background: MAIT cells are non-classically restricted T lymphocytes that recognize and rapidly respond to microbial metabolites or cytokines and have the capacity to kill bacteria-infected cells. Circulating MAIT cell numbers generally decrease in patients with active TB and HIV infection, but findings regarding functional changes differ. Methods: We conducted a cross-sectional study on the effect of HIV, TB, and HIV-associated TB (HIV-TB) on MAIT cell frequencies, activation and functional profile in a high TB endemic setting in South Africa. Blood was collected from (i) healthy controls (HC, n = 26), 24 of whom had LTBI, (ii) individuals with active TB (aTB, n = 36), (iii) individuals with HIV infection (HIV, n = 50), 37 of whom had LTBI, and (iv) individuals with HIV-associated TB (HIV-TB, n = 26). All TB participants were newly diagnosed and sampled before treatment, additional samples were also collected from 18 participants in the aTB group after 10 weeks of TB treatment. Peripheral blood mononuclear cells (PBMC) stimulated with BCG-expressing GFP (BCG-GFP) and heat-killed (HK) Mycobacterium tuberculosis (M.tb) were analyzed using flow cytometry. MAIT cells were defined as CD3+ CD161+ Vα7.2+ T cells. Results: Circulating MAIT cell frequencies were depleted in individuals with HIV infection (p = 0.009). MAIT cells showed reduced CD107a expression in aTB (p = 0.006), and reduced IFNγ expression in aTB (p < 0.001) and in HIV-TB (p < 0.001) in response to BCG-GFP stimulation. This functional impairment was coupled with a significant increase in activation (defined by HLA-DR expression) in resting MAIT cells from HIV (p < 0.001), aTB (p = 0.019), and HIV-TB (p = 0.005) patients, and higher HLA-DR expression in MAIT cells expressing IFNγ in aTB (p = 0.009) and HIV-TB (p = 0.002) after stimulation with BCG-GFP and HK-M.tb. After 10 weeks of TB treatment, there was reversion in the observed functional impairment in total MAIT cells, with increases in CD107a (p = 0.020) and IFNγ (p = 0.010) expression. Conclusions: Frequencies and functional profile of MAIT cells in response to mycobacterial stimulation are significantly decreased in HIV infected persons, active TB and HIV-associated TB, with a concomitant increase in MAIT cell activation. These alterations may reduce the capacity of MAIT cells to play a protective role in the immune response to these two pathogens.

Postnatal Expansion, Maturation, and Functionality of MR1T Cells in Humans.

MR1-restricted T (MR1T) cells are defined by their recognition of metabolite antigens presented by the monomorphic MHC class 1-related molecule, MR1, the most highly conserved MHC class I related molecule in mammalian species. Mucosal-associated invariant T (MAIT) cells are the predominant subset of MR1T cells expressing an invariant TCR α-chain, TRAV1-2. These cells comprise a T cell subset that recognizes and mediates host immune responses to a broad array of microbial pathogens, including Mycobacterium tuberculosis. Here, we sought to characterize development of circulating human MR1T cells as defined by MR1-5-OP-RU tetramer labeling and of the TRAV1-2+ MAIT cells defined by expression of TRAV1-2 and high expression of CD26 and CD161 (TRAV1-2+CD161++CD26++ cells). We analyzed postnatal expansion, maturation, and functionality of peripheral blood MR1-5-OP-RU tetramer+ MR1T cells in cohorts from three different geographic settings with different tuberculosis (TB) vaccination practices, levels of exposure to and infection with M. tuberculosis. Early after birth, frequencies of MR1-5-OP-RU tetramer+ MR1T cells increased rapidly by several fold. This coincided with the transition from a predominantly CD4+ and TRAV1-2- population in neonates, to a predominantly TRAV1-2+CD161++CD26++ CD8+ population. We also observed that tetramer+ MR1T cells that expressed TNF upon mycobacterial stimulation were very low in neonates, but increased ~10-fold in the first year of life. These functional MR1T cells in all age groups were MR1-5-OP-RU tetramer+TRAV1-2+ and highly expressed CD161 and CD26, markers that appeared to signal phenotypic and functional maturation of this cell subset. This age-associated maturation was also marked by the loss of naïve T cell markers on tetramer+ TRAV1-2+ MR1T cells more rapidly than tetramer+TRAV1-2- MR1T cells and non-MR1T cells. These data suggest that neonates have infrequent populations of MR1T cells with diverse phenotypic attributes; and that exposure to the environment rapidly and preferentially expands the MR1-5-OP-RU tetramer+TRAV1-2+ population of MR1T cells, which becomes the predominant population of functional MR1T cells early during childhood.

Osteopontin That Is Elevated in the Airways during COPD Impairs the Antibacterial Activity of Common Innate Antibiotics.

Bacterial infections of the respiratory tract contribute to exacerbations and disease progression in chronic obstructive pulmonary disease (COPD). There is also an increased risk of invasive pneumococcal disease in COPD. The underlying mechanisms are not fully understood but include impaired mucociliary clearance and structural remodeling of the airways. In addition, antimicrobial proteins that are constitutively expressed or induced during inflammatory conditions are an important part of the airway innate host defense. In the present study, we show that osteopontin (OPN), a multifunctional glycoprotein that is highly upregulated in the airways of COPD patients co-localizes with several antimicrobial proteins expressed in the airways. In vitro, OPN bound lactoferrin, secretory leukocyte peptidase inhibitor (SLPI), midkine, human beta defensin-3 (hBD-3), and thymic stromal lymphopoietin (TSLP) but showed low or no affinity for lysozyme and LL-37. Binding of OPN impaired the antibacterial activity against the important bacterial pathogens Streptococcus pneumoniae and Pseudomonas aeruginosa. Interestingly, OPN reduced lysozyme-induced killing of S. pneumoniae, a finding that could be explained by binding of OPN to the bacterial surface, thereby shielding the bacteria. A fragment of OPN generated by elastase of P. aeruginosa retained some inhibitory effect. Some antimicrobial proteins have additional functions. However, the muramidase-activity of lysozyme and the protease inhibitory function of SLPI were not affected by OPN. Taken together, OPN can contribute to the impairment of innate host defense by interfering with the function of antimicrobial proteins, thus increasing the vulnerability to acquire infections during COPD.