Antigen-specific and persistent tuberculin anergy in a cohort of pulmonary tuberculosis patients from rural Cambodia

Abstract

Purified protein derivative (PPD) skin testing is used to identify persons infected with Mycobacterium tuberculosis (Mtb) and to assess cell-mediated immune responses to Mtb. However, lack of skin induration to intradermal injection of PPD or PPD anergy is observed in a subset of patients with active tuberculosis (TB). To investigate the sensitivity and persistence of PPD reactivity and its in vitro correlates during active TB disease and after successful chemotherapy, we evaluated the distribution of skin size induration after intradermal injection of PPD among 364 pulmonary TB patients in Cambodia. A subset of 25 pulmonary TB patients who had a positive skin reaction to mumps and/or candida antigens showed persistent anergy to PPD after successful completion of TB therapy. Strikingly, in vitro stimulation of T cells from persistently anergic TB patients with mumps but not PPD resulted in T cell proliferation, and lower levels of IL-2 and IFN-gamma and higher levels of IL-10 were detected in PPD-stimulated cellular cultures from PPD-anergic as compared with PPD-reactive pulmonary TB patients. These results show that anergy to PPD is antigen-specific and persistent in a subset of immunocompetent pulmonary TB patients and is characterized by antigen-specific impaired T cell proliferative responses and a distinct pattern of cytokine production including reduced levels of IL-2.

Figure 1

Distribution of skin test reaction sizes to PPD among 364 pulmonary TB patients in Cambodia. The numbers of individuals with the indicated size of skin induration upon initial PPD screening determined within the first 2 months of initiation of TB therapy are shown in the histograms. The numbers at the top of each bar are the percentage of subjects in each size range of the total group of 364 individuals. We note that all 69 patients under the 0–4 mm range had no induration (zero).

Figure 2

T cells from anergic TB patients do not generate a PPD-specific proliferative response but can efficiently respond to mumps stimulation. CD4⁺ T cells from PPD-reactive patients (RP) and persistently PPD-anergic patients (AP) were stimulated with autologous APC loaded with PPD and mumps; [³H]thymidine incorporation (cpm) was determined 5 days later. As shown in Table 3, of the 29 persistently anergic patients, 13 were reactive to mumps and were persistently not reactive to PPD. Three of these six patients were randomly chosen, and three age- and sex-matched PPD-responsive patients were chosen and studied. The results also are presented as the mean ± SD of experiments from the three different RP and the three different AP pulmonary TB patients (all experiments performed in triplicate). The optimal concentrations of antigen loading of autologous APC (10 μg/ml for PPD or 1 μg/ml for mumps) and time length of culture (5 days) were first established in control experiments with samples from healthy PPD+ and mumps+ volunteer blood donors (data not shown). Results of three representative patients are shown.

Figure 3

Kinetics of IL-2, IFN-γ, and IL-10 production in PBMC from anergic and PPD+ TB patients. PBMC cultures were prepared from PPD-reactive patients (RP) and persistently PPD-anergic patients (AP) and were stimulated with 10 μg/ml of PPD or 1 μg/ml of mumps added directly to the bulk cultures. Supernatants were collected 0, 6, 12, 24, and 36 h later. The levels of IL-2, IFN-γ, and IL-10 at the indicated times were assessed by ELISA. Cytokine levels are depicted as the amount of secreted cytokine in PPD-stimulated cultures minus the amount in medium alone. As shown in Table 3, of the 29 persistently anergic patients, 13 were reactive to mumps and were persistently not reactive to PPD. Four of these six patients were chosen randomly, and four age- and sex-matched PPD-responsive patients were chosen and studied. The results are presented as the mean ± SE of experiments using cells from four different PPD-responding patients (RP) and four different anergic (AP) pulmonary TB patients (all experiments performed in triplicate) at each time point. We note that in previous studies, we detected the constitutive presence of IL-10-producing Tr1-like immunosuppressive T cells and a decreased number of IFN-γ-producing T cells in PBMC from PPD-anergic patients by intracellular cytokine staining (9). We were unable to detect IL-2-producing T cells by intracellular staining (data not shown). Furthermore, we did not detect IL-4, IL-12, or TGF-β protein levels in PBMC stimulated with aqueous sonicate of Mtb (H3Rv7) as measured by ELISA (9).