Detection And Frequency Distribution Of Mycobactrerial Antigen-specific CD4~+ Central Memory T Cells

Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), is one of the most important infectious disease in the world. Due to increasing migrant population, dual infection of the human immunodeficiency virus (HIV) and Mtb, and emerging of multiple-drug resistance strains of Mtb, new cases of tuberculosis (TB) are increasing worldwide. Currently, Mtb has infected approximately two billion individuals in total, with an estimated 8 million new cases, causing between 2 and 3 million deaths annually. TB has been one of the most serious public health problems. China is a high epidemic area of TB, accounted for 15% of incident cases globally, with morbidity ranking second. TB have become a heavy burden for China.Currently available vaccine against human TB is Bacilli Calmette-Guerin (BCG), an attenuated strain of M.bovis. As a live vaccine, its efficacy depends on replication inside human body after inoculation. Therefore, it does not have protective effect for adults who have been vaccinated or exposed to Mtb. Most people have been vaccinated with BCG at childhood and generated protective immune response to Mtb. Because of repeatedly transfer of culture, some important immune protective antigens of BCG had mutated, resulting in less immunoprotection.Infection rate of Mtb is high, but its morbility rate is relatively low, which indicates its occurrence, development and turnover depends not only on quantity and toxicity of Mtb, but also immune function of humans to a large extent.CD4+ T cells play an important role in the cellular immunologic response to Mtb. Initially, Mtb lived in the vacuole of macrophage. CD4+ T cells are activated and proliferated when encountering antigens presented from the mononuclear macrophage, and produce cytokines which activate the macrophage and restrain the growth of Mtb. CD4+T cells can activate, proliferate and differentiate into effector cells. After clean up of antigens, a lot of effector cells die, only a few differentiate into the memory T cells. When encountering the same antigens again, the memory T cells react fast, result in preventing or easing of the illness. This may be the reason why Mtb infected one third of the world's population, but just 5 %～10 % of them become active tuberculosis.Immunological memory plays an important role to resist TB, and provides long-term, even lifelong immune response. On account of this, we compared the production and population frequency of mycobactrerial antigen-specific CD4+ memory T cells in patients with pulmonary tuberculosis and PPD+ normal individuals, and investigated the contribution of memory cells to anti-tuberculosis immunity, offering theoretical evidence for further research.In this study, we set up a method based on CD154 expression to detect Mtb antigen-specific CD4+ memory T cells. CD154 can be used as a molecule marker for the activated antigen-specific CD4+ T cells. De novo-synthesized CD154 can identify antigen-specific CD4+ T cells in a stimulation assay. CD154 is expressed transiently on cell surface, we overcame this limitation by including fluorescently conjugated antibody to CD154 (anti-CD154) and monensin in the culture during the stimulation. Meanwhile, we proved that by flow cytometry, BCG could induce the expression of CD154 of CD4+T cells obviously.Stimulated with BCG, with surface co-staining of CD4, CD154, CCR7, and CD45RA and flow cytometry analysis, we investigated mycobactrerial antigen-specific CD4+ memory T cells and their frequency distributions in patients with pulmonary tuberculosis and PPD+ normal individuals.Twenty-three patients with pulmonary tuberculosis from a hospital in Beijing and 20 PPD+ normal individuals were examined. Compared with PPD+ normal individuals, patients with pulmonary tuberculosis had significantly reduced frequency of CD45RA-CCR7+ central memory T cells subsets (p<0.05) and elevated frequency of CD45RA+CCR7+ T cells subsets (p<0.05). The result indicates that central memory T cell subset in pulmonary tuberculosis were abnormal, which might play an important role in the development of tuberculosis and immune function.