Specific lytic activity against mycobacterial antigens is inversely correlated with the severity of tuberculosis

Abstract

The ability of peripheral blood mononuclear cells (PBMC) from patients with active tuberculosis to display cytotoxic responses against autologous Mycobacterium tuberculosis (Mtb)-pulsed macrophages was evaluated. Non-MHC restricted cell-dependent lytic activity was observed in ex vivo effector cells from tuberculosis patients and was mediated mainly by CD3(+)gammadelta TCR(+) T (gammadelta T) cells bearing CD56 and/or CD16 molecules. MHC-restricted and non-MHC restricted cytotoxic T cells (CTL) were differentially expanded upon stimulation with Mtb in tuberculosis patients and normal controls (N). Class-I restricted CD8(+) CTL and class-II restricted CD4(+) CTL were generated in PPD(+)N and to a lesser extent in PPD(-)N. Mtb-stimulated effector cells from tuberculosis patients became progressively non-MHC restricted CD4(-)CD8(-)gammadelta T cells, while lytic activity of CD4(+) and CD8(+)CTL decreased gradually as the disease became more severe. On the other hand, target cells were lysed by ex vivo cells from tuberculosis patients through the Fas-FasL and perforin pathways. Mtb-induced CD4(+) CTL from tuberculosis patients and N controls preferentially employed the Fas-FasL mechanism. Mtb-induced CD8(+) CTL effector cells from patients used the perforin-based mechanism while cells from N controls also used the Fas-FasL pathway. While Mtb-induced gammadelta CTL from patients and PPD(-)N employed the latter mechanism cells from PPD(+)N individuals also used the perforin pathway. It can be concluded that shifts in the CTL response and the cytolytic mechanisms take place as the pulmonary involvement becomes more severe.

Fig. 1

Cytotoxic effector cells present in ex vivo cells from tuberculosis patients. Ex vivo cells from TB and pre-TB patients with M or A form of the disease were incubated with anti-CD56 and anti-CD16 or anti-γδ TCR MoAb and target cells were treated with anti-MHC class-I, anti-MHC class II, anti-CD1b MoAb or isotype-matched non-relevant control antibodies before the cytotoxic assay, as described in Materials and methods. Results are expressed as percentage of cytotoxicity (mean ± s.e.m.). Statistical differences between percentage cytotoxicity from untreated effector and target cells and from MoAb-treated effector or target cells: *P < 0·05.

Fig. 2

Expression of CD16⁺, CD56⁺ on CD3^– and γδ CD3⁺ T cells present on ex vivo cells. Ex vivo cells from 15 patients with tuberculosis and eight normal controls were tested for the presence of γδ T, γδ T/CD56⁺, γδ T/CD16⁺ and CD3^-CD16⁺CD56⁺ cells, as mentioned in Materials and methods. Results are expressed as mean ± s.e.m. Statistical differences: *P < 0·05, **P < 0·01.

Fig. 3

Mtb-induced CD4⁺ αβ TCR⁺, CD8⁺ αβ TCR⁺ or γδ TCR⁺ cytotoxic effector T cells: CD4⁺ and CD8⁺ expressing the αβ TCR and CD4^–CD8^– expressing the γδ TCR receptor were obtained from 6-day cultured PBMC by negative selection with magnetic beads as described in Methods. Nineteen TB patients [nine with mild (M-TB, ▪–▪) and 10 advanced disease (A-TB, □–□)], 15 pre-TB [five with mild (M-pre-TB, ▾–▾) and 10 with advanced disease (A-pre-TB, ▿–▿] patients, five PPD⁺ N (•–•) and eight PPD⁻ N (○–○) were studied. The enriched populations were tested for their lytic activity against Mtb-pulsed macrophages in a 4-h cytotoxic assay at a 40 : 1 E/T ratio. Results are expressed as percentage of cytotoxicity (individual data). Statistical differences: CD4⁺ CTL activity − patients versus PPD⁺ N: M-TB, P < 0·05, A-TB, M-pre-TB and A-pre-TB, P < 0·002; patients versus PPD⁻ N: M-TB, P < 0·005; patients versus M-TB: A-TB, P < 0·05, M-pre-TB and A-pre-TB, P < 0·01; PPD⁺ N versus PPD− N, P < 0·002. CD8⁺CTL activity − patients versus PPD⁺ N: M-TB, P < 0·05; A-TB, M-P and A-pre-TB, P < 0·002; patients versus PPD⁻ N: A-TB, P < 0·05, M-pre-TB and A-pre-TB, P < 0·01; patients versus M-TB: A-TB, P < 0·05; M-pre-TB and A-pre-TB, P < 0·01; PPD⁺ N versus PPD⁻ N, P < 0·05. γδ CTL activity − patients versus PPD⁺ N: M-pre-TB, P < 0·05; patients versus PPD⁻ N: A-TB and M-pre-TB, P < 0·05.

Fig. 4

Correlation between activities of CTL subpopulations in patients with tuberculosis. (a) CD8⁺CTL and CD4⁺CTL; (b) γδ CTL and CD4⁺CTL. Individual CD4⁺CTL, CD8⁺CTL and γδ CTL values (% of cytotoxicity) from M-TB (▪–▪), A-TB (□–□), M-pre-TB (▾–▾) and A-pre-TB (▿–▿) patients were correlated employing the linear regression test.

Fig. 5

Lytic activity of ex vivo and Mtb-stimulated PBMC. (a) Ex vivo effector cells from six patients without previous tuberculosis (TB) [three with M (▪–▪) and three with A disease (□–□)], eight patients with a previous pulmonary tuberculosis (pre-TB) [four with M (▾–▾) and four with A disease (▿–▿)] and 13 normal individuals [(•–•), five PPD⁺ N and (○–○), eight PPD^– N] were tested for their lytic activity against autologous Mtb-pulsed macrophages (—) and non-pulsed macrophages (---) in a 4-h cytotoxic assay, employing different effector to target cell ratios (E/T) as described in Materials and methods. Results are expressed as percentage of cytotoxicity. (b) PBMC from 12 TB [five with M (▪–▪) and seven with A disease ○–○)], 10 pre-TB [four with M (▾–▾) and six with A disease (▿–▿)] patients, five PPD⁺ N (•–•) and eight PPD^– N (○–○) N controls were stimulated during 6 days with Mtb and then tested for their lytic activity as mentioned above. Results are expressed as percentage of cytotoxicity.

Fig. 6

Lytic mechanisms of Mtb-induced CD4⁺, CD8⁺ and γδ T effector cells: Mtb-stimulated CD4⁺, CD8⁺ and γδ T cells from nine M-TB, eight A-TB, eight M-pre-TB and eight A-pre-TB patients and eight PPD⁺ N and eight PPD^– N controls (effector cells, E) were incubated during 4 h with (a) Mtb-pulsed macrophages (▪); (b) anti-Fas-treated and antigen-pulsed macrophages (□); or (c) antigen-pulsed macrophages in the presence of antiperforin MoAb (). Results are expressed as percentage of lysis (× ± s.e.m.). Statistical differences between percentage lysis from (E + anti-Fas treated antigen-pulsed target cells) or from (E + antigen-pulsed target cells + antiperforin) and percentage lysis from (E + antigen-pulsed target cells): *P < 0·05, **P < 0·01.