Investigation of CD4 and CD8 T cells profiles using multicolour flow cytometry to biomarkers identification on latent tuberculosis

Abstract

The World Health Organization has established goals for eliminating tuberculosis (TB) epidemic by 2035. Tuberculosis is a contagious, airborne infectious disease, with mainly pulmonary involvement and can affect other organs. TB is a treatable disease if it is properly diagnosed. To fight TB, it is essential to search for strategies for early detection and treatment of the disease in the stages of high transmissibility. There are many difficulties in fighting against TB, including the bacillus multidrug-resistance, tuberculosis-HIV co-infection, diabetes, confinement of people (prison systems), poverty, malnutrition, and, mainly, the difficulty of diagnosing latent TB patients. For diagnosis of active (pulmonary) TB, there are diagnostic that includes the patient's anamnesis, imaging, and microbiological laboratory tests. On the other hand, latent TB is asymptomatic, but it can be diagnosed in the laboratory using tests that measure specific cellular immunity in TB, such as the tuberculin skin test and IFN-g release assays (IGRA). These assays are widely used in Public Health Policy Programs to eliminate the TB epidemic. Quantiferon-TB-Gold-Plus is universally used to diagnosis Latent TB and consists of blood collection tubes containing or not recombinant M. tuberculosis (Mtb) antigens for stimulation of T cells. Decrease of immunity difficult the detection of Mtb specific response by QuantiFERON-TB-Gold, so the search for other biomarkers, soluble, and cellular is actual and important. Several biomarkers are described in the characterization of latent TB, however, the measurement methodologies and immunological functional assays differ from laboratory to laboratory, regarding the use of reagents, design of the assay, lacking a universal standardization for comparing results more effectively. In our laboratory, we propose to use universal methodologies suggested by national and international laboratories that evaluate the immune response to Mtb, either to diagnose Latent TB or to evaluate the immunogenicity of vaccine candidates. For this purpose, we propose the standardization of stimulation of cells with immunodominant antigens present at QuantiFERON-TB-Gold-Plus assay well accepted by the scientific community and immunophenotyping of immune cells using the universal panels. In this project, we will characterize the phenotype of CD4 + T and CD8 + T cells concerning its activation, memory status, Th profile, and exhaustion in the Quantiferon-TB-Gold-Plus assay. The multiple biomarkers characterization in blood immune cells in the IGRA system can help the better diagnosis of Latent TB cases to be prevented treated as recommendations of the Ministry of Health, to avoid TB activation and transmission. (AU)