Mycobacterial antigens induce apoptosis in human purified protein derivative-specific alphabeta T lymphocytes in a concentration-dependent manner

Abstract

The morbidity and lethality of tuberculosis is partially the result of an ineffective delayed-type hypersensitivity reaction which causes caseating granulomas in the lung and other organs. Recently we showed that during caseation besides macrophages numerous Fas+ FasL+ lymphocytes undergo apoptosis and postulated that this phenomenon may be due to activation-induced cell death (AICD) as a consequence of T-lymphocyte reactivation via bacillary antigens. As purified protein derivative of Mycobacterium tuberculosis (Mtb-PPD) provokes caseation in tuberculosis patients, the question arose as to whether bacillary antigens are responsible for AICD within caseous areas. In the present study Mtb-PPD-specific T helper 1 (Th1)-differentiated T lymphocytes were generated in vitro. Reactivation of these cells with Mtb-PPD resulted in a concentration-dependent hyporesponsiveness, which was due to an increase in apoptosis of gammadelta+, alphabeta+ CD4+ as well as alphabeta+ CD8+ T lymphocytes as assessed by the demonstration of the apoptosis-associated mitochondrial membrane protein 7A6 and DNA fragmentation. Blocking experiments demonstrated that Mtb-PPD antigens exploited the Fas/FasL system to induce apoptosis in Mtb-PPD-specific T lymphocytes. These results may support the hypothesis that in tubercle granulomas with caseation T lymphocytes undergo AICD following reactivation by bacillary antigens, thus contributing to the persistence of tuberculosis.

Figure 1

Proliferation and differentiation of Mtb-PPD-specific T lymphocytes. Human lymphocytes were cultured with autologous DC and Mtb-PPD (autologous MLC). After 6 days, uptake of [³H]thymidine and release of Th1/Th2-associated cytokines were measured. The results of a representative autologous MLC are expressed as mean±SD of three replicates. The [³H]thymidine assay shows high proliferative activity in MLC when compared to controls (a). Cytokine release assays demonstrate that in MLC the production and secretion of IFN-γ, (b) but not of IL-5 or (c) of IL-10 (d) are increased.

Figure 2

Characterization of Mtb-PPD-specific T lymphocytes. Human lymphocytes were cultured with autologous DC and Mtb-PPD (autologous MLC) for 6 days. Two-colour dot plot analysis of a representative autologous MLC shows that in the course of culturing the percentage of αβ⁺ CD4⁺, αβ⁺ CD8⁺, and γδ⁺ CD4^– CD8^– cells shifts in favour of αβ⁺ CD8⁺ cells (left panel), and the expression of the Fas-receptor and its ligand FasL increases in MLC-derived CD3⁺ T lymphocytes when compared to peripheral blood lymphocytes (right panel).

Figure 3

Proliferation of Mtb-PPD-specific T lymphocytes following reactivation. Human lymphocytes were cultured with autologous DC and Mtb-PPD (autologous MLC) for 6 days. Following separation from MLC sensitized T lymphocytes were reactivated by different concentrations of Mtb-PPD in the absence of DC. The proliferative response of reactivated lymphocytes were determined by applying [³H]-thymidine assay (a). As control Mtb-PPD-specific T lymphocytes were reactivated by DC pulsed with the same concentrations of Mtb-PPD (b). The results of a representative experiment with lymphocytes from a donor are expressed as c.p.m. ± SD of three replicates. Evidence shows that in the absence of DC the more the Mtb-PPD concentration the less the proliferative rate of reactivated lymphocytes. Conversely in the presence of DC Mtb-PPD does not affect the proliferation activity of lymphocytes.

Figure 4

Apoptosis of Mtb-PPD-specific T lymphocytes following reactivation. Human lymphocytes were cultured with autologous DC and Mtb-PPD (autologous MLC) for 6 days. Following separation from MLC sensitized T lymphocytes were reactivated by different concentrations of Mtb-PPD. The apoptotic rate of reactivated lymphocytes were determined by flow cytometric analysis of the apoptosis-associated mitochondrial membrane protein 7A6 (left panel) and TUNEL (right panel). Two-colour dot plots of a representative experiment with lymphocytes from a donor indicate that the apoptotic rate of reactivated CD3⁺ lymphocytes is positively correlated with increasing Mtb-PPD concentrations.

Figure 5

Characterization of reactivated T lymphocytes undergoing apoptosis. Mtb-PPD-specific human T lymphocytes were reactivated by 400 µg/ml Mtb-PPD. To characterise apoptotic T-cell populations, the TUNEL technique was combined with cell surface staining for different T-cell markers. Two-colour dot plots show a representative experiment with lymphocytes from a donor following reactivation by 400 µg/ml Mtb-PPD. Results indicate that CD3⁺, CD4⁺, CD8⁺, αβ⁺ as well as γδ⁺ cells undergo apoptosis.

Figure 6

Effect of Fas on re-call response of reactivated Mtb-PPD-specific T lymphocytes. Mtb-PPD-specific human T lymphocytes were reactivated by different concentrations of Mtb-PPD. The death receptor Fas was blocked on Mtb-PPD-specific lymphocytes by a neutralizing anti-Fas antibody (a and b, white bars). As control irrelevant mouse IgG was added to the cultures (a, b, black bars). The proliferative and apoptotic rates of reactivated lymphocytes were determined by applying [³H]thymidine assay (a) and TUNEL (b), respectively. The results of a representative experiment with lymphocytes from a donor are expressed as ±SD of three replicates. Evidence demonstrates that following Fas-blockade the proliferative rate of Mtb-PPD-specific lymphocytes reactivated by 100 or 400 µg/ml Mtb-PPD increases (a, white bars), when compared to the controls (a, black bars) (P < 0·05). Conversely Fas-blockade leads to a decrease in apoptotic rate of Mtb-PPD-specific reactivated by 100 or 400 µg/ml Mtb-PPD (b, white bars), when compared to the controls (b, black bars) (P < 0·05).