Activation of MAIT cells by antigen presenting cells: a comprehensive analysis and assessment of HIV and TB disease on these interactions

MAIT cells are non-classical, innate-like T lymphocyte subsets, which recognize microbial vitamin B metabolites and rapidly respond by producing pro-inflammatory cytokines such as IFN-γ, TNF-α and cytotoxic molecules, which may result in the killing of bacteria-infected cells. Unlike conventional T cells, MAIT cells can be activated by either antigen presentation on MR1 or directly by cytokines. MAIT cells play a protective role against bacterial infections in mice but so far, no human studies have confirmed a direct role of MAIT cells in the control of bacterial infections or prevention of disease progression. Circulating MAIT cell numbers decrease in patients with active TB, but findings regarding functional changes have been conflicting. The aims of this study were to assess the cellular interactions between antigen presenting cells and MAIT cells, and determine the effect of TB, HIV and HIV-associated TB on MAIT cell numbers, activation, inhibitory and functional profile. We recruited 26 healthy controls (HC) without HIV or TB disease, 30 people with HIV only, 30 with active TB only, and 26 with HIV-associated TB. All TB patient samples were obtained before treatment. Blood was collected from all participants and peripheral blood mononuclear cells (PBMC) isolated from the blood and cryopreserved. Later, PBMC were thawed, rested and stimulated with BCG expressing GFP (BCG-GFP) and heat-killed (HK) M.tb. Media only, LPS and PHA were used as stimulation controls. The numbers, functional profile, inhibitory and activation status of MAIT cells were determined by flow cytometry. Soluble cytokines were measured by ELISA and multiplex Luminex assays. For HIV-infected participants with no TB and patients with HIV-associated TB, the median CD4 counts were 501 cells/µL and 228 cells/µL, and HIV viral loads were 1673 copies/mL and 66509 copies/mL, respectively. 63% and 69% of HIV infected patients were on ART in the HIV alone and HIV/TB groups, respectively. We observed lower frequencies of circulating MAIT cells in individuals with active TB only or HIV only compared to HC. HIV/TB resulted in lower but nonsignificant MAIT cell frequencies and numbers compared to HC. In response to stimulation with whole mycobacteria, MAIT cells were more highly activated (expressing high HLA-DR) in people with TB and HIV-associated TB compared to HC. Furthermore, MAIT cells from people with active TB only had significantly upregulated PD-1 expression compared to HC. MAIT cells from individuals with active TB and HIV-associated TB had a lower capacity to degranulate (express CD107a) and produce IFN-γ compared to HC. HIV-infection alone did not affect these functions. The levels of soluble IFN-α2 were reduced in the groups with HIV only and active TB only while IFN-γ was reduced in all patient groups. Blocking experiments revealed that MAIT cell activation in response to BCG was primarily through MR1 antigen presentation pathway. The level of monocyte and dendritic cell infection (expression of GFP) was similar in all groups. We observed a positive correlation between monocyte infection and the frequencies of MAIT cells producing IFN-γ in people with active TB only. We also observed a positive correlation between the amount of soluble IL-12 and the proportion of MAIT cells producing IFN-γ and CD107a in HC and in people with HIV infection, respectively. Our data show that HIV, TB and or HIV/TB result in a decrease in circulating MAIT cells, impaired functional profile, and a significant alteration in activation status and inhibitory potential. The MR1 antigen presentation was the predominant pathway for MAIT cell activation. There was also a relationship observed between MAIT cell activation and magnitude of innate response to mycobacterial antigens. These results provide further evidence of the potential role of MAIT cells in infectious disease pathogenesis and demonstrate how HIV and TB alter their function

Mycobacterial-specific secretion of cytokines and chemokines in healthcare workers with apparent resistance to infection with Mycobacterium tuberculosis

BackgroundCurrently, diagnosis of latent TB infection (LTBI) is based on the secretion of IFN-γ in response to Mycobacterium tuberculosis (Mtb) antigens, the absence of which is regarded as no infection. Some individuals appear to resist Mtb infection despite sustained exposure (resisters). In this study, we aimed to assess cytokines, chemokines and antibodies that may be associated with resistance to Mtb infection. We hypothesized that there may be an alternative immune response to Mtb exposure in the absence of IFN-γ in resisters.MethodsWe enrolled HIV-uninfected healthcare workers who had worked in high TB-exposure environments for 5 years or longer. We screened them for LTBI using the tuberculin skin test and the QuantiFERON-TB Gold Plus assay. We performed multiplex Luminex to measure concentrations of T cell-associated cytokines and chemokines as well as total antibodies in plasma collected from unstimulated fresh whole blood and supernatants from QuantiFERON-TB Gold Plus tubes following incubation of whole blood for 16-24 hours with ESAT6/CFP10 peptides.ResultsSamples from 78 individuals were analyzed: 33 resisters (TST<10mm; IGRA<0.35 IU/mL), 33 with LTBI (TST≥10mm and IGRA≥0.35 IU/mL) and 12 discordant (TST=0mm; IGRA≥1.0 IU/mL). There were no differences in concentrations of cytokines and chemokines in plasma between the different groups. Resisters had significantly lower concentrations of IFN-γ, IL-2, TNF-α, MIP-1α, MIP-1β, ITAC, IL-13 and GM-CSF in supernatants compared with LTBI group. There were no significant differences in the concentrations in supernatants of IL-10, IL-1β, IL-17A, IL-21, IL-23, MIP-3α, IL-4, IL-5, IL-6, IL-7, IL-8, Fractalkine and IL-12p70 between the groups. We observed that resisters had similar concentrations of total antibodies (IgG1, IgG2, IgG3, IgG4, IgA, and IgM) in plasma and supernatants compared to the LTBI and discordant groups.ConclusionResistance to Mtb infection despite sustained exposure is associated with lower Mtb-specific secretion of Th1-associated cytokines and chemokines. However, resisters showed secreted concentrations after Mtb stimulation of total antibodies and cytokines/chemokines associated with innate and Th17 immune responses similar to those with Mtb infection. This suggests an ability to mount non-IFN-γ immune responses to Mtb in apparent resisters

Serial measurement of M. tuberculosis in blood from critically-ill patients with HIV-associated tuberculosis

BACKGROUND: Despite being highly prevalent in hospitalised patients with severe HIV-associated tuberculosis (TB) and sepsis, little is known about the mycobacteriology of Mycobacterium tuberculosis bloodstream infection (MTBBSI). We developed methods to serially measure bacillary load in blood and used these to characterise MTBBSI response to anti-TB therapy (ATT) and relationship with mortality. METHODS: We established a microscopy method for direct visualisation of M. tuberculosis bacilli in blood using a novel lysis-concentration protocol and the fluorescent probe, 4-N,N-dimethylaminonaphthalimide-trehalose (DMN-Tre). We tested blood using GeneXpert® MTB/RIF-Ultra (Xpert-ultra) and Myco/F lytic culture after processing blood through lysis-wash steps to remove PCR inhibitors and anti-microbial drug carry-over. HIV-positive patients predicted to have MTBBSI gave blood samples 0, 4, 24, 48 and 72 h after ATT initiation. Bacillary loads were quantified using microscopy, Xpert-ultra cycle threshold, and culture time-to-positivity. Pharmacodynamics were modelled using these measures combined on an ordinal scale, including association with 12-week mortality. FINDINGS: M. tuberculosis was detected in 27 of 28 recruited participants; 25 (89%) by blood Xpert-ultra, 22 (79%) by DMN-Tre microscopy, and 21 (75%) by Myco/F lytic blood culture. Eight (29%) participants died by 12-week follow-up. In a combined pharmacodynamic model, predicted probabilities of negative DMN-Tre microscopy, blood Xpert-ultra, or blood culture after 72 h treatment were 0·64, 0·27, and 0·94, respectively, in those who survived, compared with 0·23, 0·06, and 0·71 in those who died (posterior probability of slower clearance of MTBBSI in those that died >0·99). DMN-Tre microscopy of blood demonstrated heterogenous bacillary morphologies, including microcolonies and clumps. Bacillary cell-length varied significantly with ATT exposure (mean cell-length increase 0·13 log-µm/day; 95%CrI 0·10–0·16). INTERPRETATION: Pharmacodynamics of MTBBSI treatment can be captured using DMN-Tre microscopy, blood Xpert-ultra and culture. This could facilitate interventional trials in severe HIV-associated TB. FUNDING: Wellcome Trust, NIH Fogarty International Center, South African MRC, NIHR(UK), National Research Foundation of South Africa