Mycobacterium tuberculosis infected macrophages lead to apoptosis of antigen activated CD8 T cells

Abstract

Aims and objectives The role of CD4 T cells in immuno-pathogenesis of Mycobacterium tuberculosis (MTB) has been demonstrated in mouse models and in humans. However, the role of CD8 T cells in protective immunity against TB needs elucidation. Depletion of cytotoxic CD8 T cells have been found to be associated with reduced immunity to TB in different animal models. Apoptosis and hypo-responsiveness of T cells have been reported in TB patients and correlated with the persistence of infection. In a previous report, MTB specific T cells were observed, as well as bystander T cells’ apoptosis, which is induced by ex vivo infected autologous macrophages. It becomes relevant to find out the effect of infected macrophages on antigen-specific CD8 T cells during infection. Method Peripheral blood mononuclear cells (PBMC) were isolated by density gradient centrifugation from 10 healthy BCG-vaccinated subjects. Monocytes from one aliquot of PBMC matured to macrophages for five days. These macrophages were infected with H37Rv at MOI of 1:10. In parallel, PBMC from a second aliquot were stimulated with CFP/WCL at a concentration of 5 μg/ml in the presence of IL-2 for five days. To study whether cytotoxic CD8 T cells undergo apoptosis in the presence of MTB-infected macrophages, CFP/WCL activated T cells were co-cultured with infected macrophages and uninfected macrophages for a further five days. The parameters studied were the expression of AnnexinV as an apoptosis marker, expression of CD95 to understand the role of Fas-FasL mediated apoptosis, expression of CD45/RO as a memory cell marker, expression of CD25 as activation markers on CD4 and CD8 T cells through flow cytometry at 48 h and 5 days of co-culture. The levels of IFN-γ and TNF-α were studied in the respective supernatant. Results As compared with unactivated PBMC a significantly high percentage of CD4/CD25+ and CD8/CD25+ cells in infected Mφ-CFP/WCL stimulated T cell culture indicate the activation of T cells in response to MTB antigens. A significant percentage of CFP/WCL activated CD8 T cells were found to be AV positive in the infected co-culture assay at the fifth day indicating the apoptosis of antigen activated CD8 T cells. Interestingly, a significant percentage of CFP/WCL activated CD8/RO+ memory cells were found at 48 h of co-culture, but this percentage was reduced at the fifth day of co-culture. However, the CD4/RO cells percentage remained constant until the fifth day of co-culture. This reduction in CD8 /RO cells and the increase in CD8/AV+ cells suggested apoptosis of memory cells which may be induced by infected macrophages. A comparable percentage of CD8/CD95+ cells in infected and uninfected co-culture wells at both 48 h and the fifth day was observed. As compared with unactivated PBMC, the percentage of CD8/CD95+ cells was found to be significantly higher in both infected and uninfected co-culture assays, suggesting that observed increased apoptosis in infected co-culture assays was not mediated through Fas-FasL pathway. In the infected co-culture supernatant the IFN-γ concentration declined by the fifth day as compared with the uninfected co-culture; this can be correlated with reducing T cell number during infection. In contrast, a positive correlation was observed between the concentration of TNF-α and the percentage of CD8/AV+ cells by the fifth day in infected co-culture. It indicated the role of TNF-α released by MTB-infected macrophages in mediating CD8 T cell apoptosis. Conclusion The present data indicates that MTB infection of macrophages could be responsible for apoptosis of CD8 T cells, which may be correlated with impaired immunity against TB.