Tuberculosis caused by Mycobacterium tuberculosis (MTb) continues to represent a major public health problem worldwide and it is the main cause of death among people living with HIV/AIDS. Mycobacteria-specific CD4 T cells play an important role in the control of mycobacterial infection and seem to be impaired in HIV patients, including those receiving highly active antiretroviral therapy. The aim of this project was to establish and use in vitro assays to characterize mycobacteria-specific effector T cell functions in HIV-1 infected patients in comparison with healthy controls.
For establishing the methods, blood samples from healthy donors were used and peripheral blood mononuclear cells (PBMCs) isolated by density gradient centrifugation. To analyze frequencies of mycobacteria-specific T cells in peripheral blood, PBMCs were stimulated overnight with different mycobacterial antigens. After intracellular staining, effector cytokine production (IFN-γ, TNF and IL-2) from T cells was analyzed by multicolor flow-cytometry.
Viability and mycobacteria-specific T cell effector cytokine production was assessed in fresh cells as well as after cryopreservation. Mycobacteria-specific T cells were found to occur at very low frequencies in peripheral blood. Therefore a protocol was established to expand mycobacteria-specific T cells in vitro and to analyze mycobacteria-specific effector T cell functions in a human antigen-specific setting. These enriched mycobacteria-specific T cells were used to analyze kinetics of T cell effector cytokine production in response to stimulation with infected macrophages. In addition, antigen-expanded T cells were used to analyze the inhibitory effect of T cells on growth of mycobacteria in macrophages.
Another aim of this project was to analyze frequencies and functionality of mycobacteria-specific T cells in blood samples from HIV-1 patients and healthy controls. Therefore the protocol to quantify CD4 effector T cell subsets was optimized to give low background and allow sensitive and reproducible T cell analysis directly from isolated blood cells. Stimulation was tested with live Mycobacterium avium (M. avium), causing opportunistic infections in HIV-patients, as well as purified antigens from MTb and M. avium. At the end of the project, samples from two HIV patients were analyzed. Results pointed towards an impaired mycobacteria-specific immune response in the HIV patients with differences in polyfunctionality of the T cell responses. These preliminary findings encourage the use of the technique in further studies comparing T cell responses towards MTb and M. avium antigens as well as live M. avium in healthy donors and HIV patients at different stages of infection.
The tools established in this study are open for a variety of different analyses such as studies with different antigens, modified macrophages or modified mycobacteria and might contribute to a better understanding of still incompletely understood mechanisms involved in anti-mycobacterial immunity.
2012. , 78 p.