Loss of receptor on tuberculin-reactive T-cells marks active pulmonary tuberculosis

Abstract

Background: Tuberculin-specific T-cell responses have low diagnostic specificity in BCG vaccinated populations. While subunit-antigen (e.g. ESAT-6, CFP-10) based tests are useful for diagnosing latent tuberculosis infection, there is no reliable immunological test for active pulmonary tuberculosis. Notably, all existing immunological tuberculosis-tests are based on T-cell response size, whereas the diagnostic potential of T-cell response quality has never been explored. This includes surface marker expression and functionality of mycobacterial antigen specific T-cells.

Methodology/principal findings: Flow-cytometry was used to examine over-night antigen-stimulated T-cells from tuberculosis patients and controls. Tuberculin and/or the relatively M. tuberculosis specific ESAT-6 protein were used as stimulants. A set of classic surface markers of T-cell naïve/memory differentiation was selected and IFN-gamma production was used to identify T-cells recognizing these antigens. The percentage of tuberculin-specific T-helper-cells lacking the surface receptor CD27, a state associated with advanced differentiation, varied considerably between individuals (from less than 5% to more than 95%). Healthy BCG vaccinated individuals had significantly fewer CD27-negative tuberculin-reactive CD4 T-cells than patients with smear and/or culture positive pulmonary tuberculosis, discriminating these groups with high sensitivity and specificity, whereas individuals with latent tuberculosis infection exhibited levels in between.

Conclusions/significance: Smear and/or culture positive pulmonary tuberculosis can be diagnosed by a rapid and reliable immunological test based on the distribution of CD27 expression on peripheral blood tuberculin specific T-cells. This test works very well even in a BCG vaccinated population. It is simple and will be of great utility in situations where sputum specimens are difficult to obtain or sputum-smear is negative. It will also help avoid unnecessary hospitalization and patient isolation.

Figure 1. Flow diagram of the blind prospective diagnostic study.

Eligible patients were recruited by the admitting physician. Diagnostic standard refers to the standard diagnostic procedure for TB in this hospital including history, X-ray, sputum microscopy and culture, PCR, biopsy and histology where applicable. TST = tuberculin skin test

Figure 2. Distribution of tuberculin-reactive CD4 T-cells among the subsets defined by classic memory/naïve markers.

PBMC were stained with the indicated monoclonal antibodies. Panel A shows CD3 T-cells, panels B-D only CD4 T-cells. IFN-γ-producing ex-vivo tuberculin-reactive events are highlighted black. The dotted horizontal line in B shows the limit set for CD27-positivity. Representative results from one patient with active TB are shown.

Figure 3. Significantly fewer tuberculin-reactive CD4 T-cells express CD27 in TB-patients than in controls.

Proportions of CD27-positive and negative cells were based on a minimum of 50 IFN-γ positive events. Vertical numbers indicate evaluated events (median and range). Controls included unexposed controls, professionally TB-exposed health care workers, and donors with latent infection. A threshold of 49% would effectively discriminate between patients and controls (dotted line). Controls with latent TB infection had higher values than individuals with no known exposure to TB.

Figure 4. Loss of CD27 on tuberculin-reactive CD4 T-cells identifies smear and/or culture positive TB.

A: Patients with smear and/or culture positive pulmonary TB were distinguishable from those with smear and culture negative pulmonary TB using a threshold of 48% (dotted line). Patients more than 1 year after beginning of treatment mostly had values >48% if TB was initially smear positive (empty squares), and <48%, if TB was smear negative (filled squares). Statistically significant differences are indicated (Chi-square test). Primary end-point was the percentage of CD27-negative cells among reactive cells. Vertical numbers indicate the number of evaluated events (median and range). B: Loss of CD27 expression on tuberculin and ESAT-6 specific CD4 T-cells is closely correlated.

Figure 5. CD27 expression on tuberculin-reactive CD4 T-cells reverts very slowly .

A: CD27-expression on tuberculin-reactive CD4 T-cells was plotted against the length of the interval between the collection date of the analyzed blood sample and the collection date of the last positive sputum sample (all data from blinded study). Negative values indicate that sputum was already smear and culture negative when tuberculin-reactive CD4 T-cells were analyzed, positive values indicate that sputum samples were still positive and remained so for the indicated time. B: serial measurements in 7 individuals (data analysis blinded).