Conceived and designed the experiments: HV FK MS LT TU RL HM. Performed the experiments: FK MS LT VY AY. Analyzed the data: FK MS LT VY. Contributed reagents/materials/analysis tools: FK RL AQ GL LN HM. Wrote the paper: HV FK MS LT TU LN. Other: Designed the study: FK. Patient recruitment, clinical assessment, grouping of patients: GL AQ. Wrote the first draft of the paper: FK.
The authors have declared that no competing interests exist.
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
Flow-cytometry was used to examine over-night antigen-stimulated T-cells from tuberculosis patients and controls. Tuberculin and/or the relatively
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.
The goal of this work was to establish a new biomarker for clinical tuberculosis based on measurable changes that TB specific CD4 T-cells undergo as they differentiate in response to virulent
The role of T-cells in controlling TB is dramatically illustrated by the HIV pandemic. Whereas only 10% of latently TB infected but otherwise healthy individuals progress to active TB in their lifetime, this figure increases to 10% per year in HIV infected individuals
Since CD4 T-cells are so important in the defence against TB, we were hoping to find differences in the degree of differentiation resulting from the ‘one-off’ stimulus provided by BCG vaccination on the one hand and the exposure to virulent replicating
Patterns of antigen-dependent T-cell differentiation have been a central issue in T-cell immunology for a number of years both in humans and animal models
CD27 is a receptor for co-stimulation expressed on the T-cell surface and down-regulated when T-cells progress from a non-antigen experienced towards a terminal memory stage. It was recently shown that IFN-γ producing CD4 T-cells in the lungs of TB infected mice are predominantly CD27-negative
Blood from more than 30 patients with TB at different stages of treatment was used to establish antibody staining panels using tuberculin and/or ESAT-6 for stimulation. An initial comparative and retrospective study focused on the distribution of CD27 on tuberculin-reactive CD4 T-cells and included 6 patients with open pulmonary TB (4 female, 2 male, age 23–79 years) and 31 healthy individuals with no signs or symptoms of TB but various degrees of exposure (household contacts, health care workers in a lung hospital, medical students, and unexposed healthy blood donors; 23 female, 8 male, age 22–69 years). Within this group 5 individuals had latent TB infection, 17 were highly exposed, and 9 were considered unexposed. The diagnosis of latent TB infection was established by a positive response to tuberculin in non-BCG vaccinated individuals (n = 3). Two individuals who had shared their bed with a patient with smear positive TB where also classified as having latent TB. In individuals classified as ‘highly exposed’ latent TB infection had not been conclusively demonstrated but for some of them would be suggested by their very close and continued exposure. These included hospital personnel with regular direct contact with TB, including some regularly performing BAL on TB patients.
A blinded prospective study was designed to confirm the results of the initial study. The diagnoses relating to coded samples were not revealed to the lab until results had been reported back in print to the clinicians obtaining and coding the samples. For this study 31 additional patients with smear and/or culture positive or smear and culture negative pulmonary TB (11 female, 20 male, median age 51 years, range 15–96 years) were recruited within two months of treatment initiation, 9 patients at follow-up at least one year after beginning of therapy, and 20 control patients with no known history of or exposure to TB (6 female, 14 male, age 40–76 years). Disease severity in TB patients was based on the number and size of lesions observed on X-ray, however, these data were not used to subgroup patients. All TB patients except for one had more than one lesion on X-ray with at least one lesion larger than 3 cm diameter. Treatment was based on triple (isoniazid, ethambutol, and rifampicin) or quadruple (triple plus pyrazinamide) regimen. It was continued for one year in all patients and prolonged depending on follow-up evaluation. In most cases, TB culture results were pending at the time of sample collection for flow-cytometry whereas a sputum smear had been obtained. Blood was drawn into citrated blood collection tubes following written informed consent as approved by the Charité Ethics Committee. The study protocol was in accordance with the Declaration of Helsinki. A flow diagram of the study is presented in
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
This method was described in detail elsewhere
Pacific-blue (PB)-labeled anti-CD3, AmCyan-labeled anti-CD4, Fluorescein-Iso-Thio-Cyanate (FITC)-labeled anti-IL2, anti-CD27, and anti-CD28, Phycoerythrin(PE)-labeled anti-IL-2 and anti-CD62L, Peridine-Chlorophyll(PerCP)-labeled anti-CD3, Allophyocyanine(APC)-labeled anti-IFN-γ were purchased from BD. PE-Texas-Red-(ECD)-labeled anti-CD4 and Phycoerythrin(PE)-labeled anti-CD57 where purchased from Beckman-Coulter (Krefeld, Germany), PE-Cy7-labeled anti-CD8 was purchased from Caltag (Hamburg, Germany). Fluorochrome-labeled isotype controls were used as appropriate.
Flow-cytometry was performed on an LSR II flow-cytometer (BD). At least 250,000 lymphocytes were acquired per tube. Data files were analyzed using FACSDiva™ software (BD). The percentage of IFN-γ-positive CD4 T-cells was determined by a standardized strategy. The median number of positive events evaluated was 479. Given 479 events the 95%-confidence interval for a proportion of CD27-negative events determined to be 50% would be 50+/− 4.5% (given normal distribution). Distributions based on a minimum of 50 and 20 events were considered for tuberculin and ESAT-6 stimulated samples, respectively, the latter only when comparing the distribution of tuberculin and ESAT-6 specific CD4 T-cells. Whether more than 50 tuberculin-reactive CD4 T-cells could be collected depended on the patient lymphocyte count, reactivity, and the number of stimuli and antibody panels tested.
The ESAT-6 peptide set was made in-house (21 peptides, 15 amino-acid length, 11 overlaps between peptides, covering the Swiss-Prot Q57165 sequence). Peptides were dissolved in DMSO and used as described above. Tuberculin (PPD) was purchased from SSI (Copenhagen, Denmark).
Supernatants of overnight stimulated samples (same conditions as above save addition of Brefeldin A) were stored at −80°C until measurement using a custom ordered bead-array (Lincoplex™) by Linco Research (Stockholm, Sweden) and a Luminex-xMAP System.
The reference laboratory tests (smear and culture) were performed by fully trained hospital and laboratory personnel (routine TB diagnostic lab). Biopsies were analysed by a fully trained experienced pathologist. X-rays were evaluated by fully trained and experienced chest physicians/radiologists. All samples were prepared and measured by 2 laboratory scientists with long-term experience in flow-cytometry. Flow-cytometric data analysis was performed by a highly experienced medical scientist.
SPSS 11.0 (SPSS Inc, Chicago, USA) software was used. Differences between paired samples were analysed using the Wilcoxon test. Receiver Operating Characteristic (ROC)-analysis was used to define cut-off values. Differences between unpaired sample were tested using the Mann-Whitney U-test.
The expression of “classic” markers of T-cell differentiation
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.
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.
To be statistically viable, results shown in
To further explore the possibility of diagnosing TB based on the CD27-expression of TB specific T-helper-cells, a prospective
Dx | code | Age (years) | sex | TST (mm) | Tuberculin stimulated (10 µg per ml) | ESAT-6 stimulated (20 µg of peptide per ml) | ||
CD4 T-cells (%) | IFN-γ (IU) | CD4 T-cells (%) | FN-γ(IU) | |||||
1 | B1 | 45.94 | m | 6 | 0.1430 | 4.90 | 0.0158 | 0.15 |
1 | B3 | 81.57 | f | 10 | 0.2962 | 1.33 | 0.0000 | nd |
1 | B11 | 32.96 | m | nd | 0.1833 | 3.77 | 0.0066 | 0.00 |
1 | B12 | 50.63 | f | 10 | 0.1298 | 4.00 | 0.0029 | 0.00 |
1 | B33 | 31.85 | f | nd | 0.0143 | 0.69 | 0.0010 | 0.00 |
1 | B48 | 66.23 | m | nd | 0.0740 | 1.42 | 0.0005 | 0.00 |
1 | B49 | 14.99 | m | <5 | 0.1031 | 10.59 | 0.0007 | 0.00 |
1 | B52 | 71.11 | m | <5 | 0.1374 | 1.10 | 0.21 | |
1 | B61 | 28.51 | m | nd | 0.1411 | 9.03 | 1.46 | |
1 | B75 | 75.85 | f | 35 | 0.3035 | 10.23 | Nd | 1.00 |
1 | B76 | 95.69 | m | nd | 1.2005 | 4.08 | 0.01 | |
1 | B77 | 28.64 | f | 5 | 0.8713 | 44.79 | 1.83 | |
Patients with smear and culture negative pulmonary TB | ||||||||
3 | B21 | 35.33 | f | 20 | 0.1405 | 1.99 | 0.0023 | 0.00 |
2 | B29 | 69.58 | m | <5 | 0.0012 | 0.00 | 0.0013 | 0.00 |
2 | B40 | 50.41 | m | 10 | 0.3424 | 12.89 | 0.0003 | 0.00 |
2 | B42 | 41.97 | m | 28 | 0.1359 | 5.86 | 0.0005 | 0.00 |
4 | B46 | 71.33 | m | 30 | 0.5766 | 13.37 | 0.69 | |
2 | B47 | 66.7 | m | <5 | 0.0200 | 0.04 | 0.0043 | 0.00 |
2 | B59 | 64.73 | m | 5 | 0.0926 | 0.43 | 0.0012 | 0.00 |
2 | B65 | 74.38 | m | nd | 0.0034 | 0.00 | 0.0049 | 0.00 |
2 | B70 | 70.99 | f | <5 | 0.0026 | 0.03 | 0.0016 | 0.00 |
2 | B73 | 15.82 | m | 10 | 0.0145 | 0.00 | 0.0046 | 0.00 |
Patients with pulmonary TB at least one year after beginning of treatment | ||||||||
2 | B7 | 21.22 | m | 12 | 0.1996 | 6.59 | 0.41 | |
1 | B22 | 50.24 | f | nd | 0.3246 | 16.66 | 0.0023 | 0.12 |
1 | B27 | 58.56 | m | 16 | 0.2539 | 7.79 | 0.0018 | 0.00 |
1 | B43 | 76.38 | f | 12 | 0.7997 | 3.23 | 0.0049 | 0.01 |
1 | B51 | 36.46 | m | nd | 0.0221 | nd | 0.0222 | nd |
1 | B56 | 61.23 | f | <5 | 0.0417 | 0.24 | 0.15 | |
1 | B57 | 60.11 | m | <5 | 0.0788 | 2.18 | 0.0006 | 0.00 |
1 | B58 | 37.47 | f | 25 | 0.0308 | 2.41 | 0.26 | |
1 | B60 | 49.69 | m | 5 | 0.0764 | 2.59 | 0.0009 | 0.00 |
Control patients | ||||||||
6 | B8 | 44.68 | f | <5 | 0.0151 | 0.00 | 0.0039 | 0.00 |
6 | B10 | 58.92 | m | <5 | 0.0471 | 0.00 | 0.00 | |
6 | B15 | 71.3 | m | <5 | 0.0252 | 0.01 | 0.0001 | nd |
6 | B16 | 55.11 | f | nd | 0.0022 | 0.02 | nd | 0.00 |
6 | B17 | 40.84 | m | <5 | 0.1161 | 1.00 | 0.0030 | 0.00 |
6 | B18 | 65.85 | f | nd | 0.0613 | 0.25 | nd | |
6 | B23 | 70.01 | m | <5 | 0.2552 | 2.55 | 0.0060 | 0.00 |
6 | B24 | 42.35 | m | <5 | 0.0273 | 0.02 | 0.0046 | 0.00 |
6 | B25 | 76.07 | f | <5 | 0.0255 | 0.00 | 0.0012 | 0.00 |
6 | B26 | 69.91 | f | nd | 0.0086 | 0.00 | 0.0000 | 7.12 |
6 | B28 | 64.3 | m | <5 | 0.0078 | 0.23 | 0.0007 | 0.00 |
6 | B35 | 48.05 | m | <5 | 0.0058 | 0.27 | 0.0013 | 0.00 |
6 | B36 | 58.67 | m | nd | 0.0088 | 0.16 | 0.0063 | 0.00 |
6 | B39 | 47.24 | m | <5 | 0.3653 | 6.06 | 0.0040 | 0.72 |
6 | B41 | 43.94 | m | <5 | 0.0019 | 0.18 | 0.0005 | 0.00 |
6 | B44 | 73.7 | m | nd | 0.1972 | 0.50 | 0.13 | |
6 | B55 | 54.19 | m | <5 | 0.0035 | 0.01 | 0.0017 | 0.00 |
mainstay of diagnosis: 1 = positive culture and/or microscopy, 2 = mycobacteria in biopsy, 3 = therapy response, 4 = typical chest film, TST >15 mm, positive response to ESAT-6
results exceeding 1 positive event per 10,000 cells were counted as positive and are shown in bold
some of these results exceed the threshold for positivity published for a commercial assay (Quantiferon Gold, Cellestis) of 0.35 IU.
The blind study confirmed the findings of the initial study (
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.
Group | CD27-neg. tuberculin reactive CD4 T-cells | Total | |
< 48% | >48% | ||
1 | 0 | 11 | 11 |
2 | 6 | 0 | 6 |
3 | 3 | 3 | 6 |
4 | 8 | 0 | 8 |
Total | 17 | 14 | 31 |
As in the initial study, reactivity to tuberculin measured either by IFN-γ in the supernatant or the frequency of tuberculin-reactive CD4 T-cells would not have been able to discriminate the groups (see
Patient group | TST≥5 mm | tuberculin in vitro | ESAT-6 in vitro |
smear and/or culture positive TB (n = 12) | 5/7 | 12/12 vs. 12/12 | 5/11 vs. 5/11 |
smear and culture negative TB (n = 10) | 6/9 | 7/10 vs. 5/10 | 1/10 vs. 1/10 |
≥1 year after beginning of TB treatment (n = 8) | 5/7 | 9/9 vs. 8/8 | 4/9 vs. 4/8 |
Control patients (n = 17) | 0/12 | 10/17 vs. 9/17 | 3/16 vs. 3/15 |
not all test were performed in all individuals, numbers refer to positive test results per tested individuals
frequency of tuberculin-reactive CD4 T-cells (IFN-gamma) above 1/10,000 or IFN-gamma above 0.1 IU/ml (arbitrary value)
frequency of ESAT-6 reactive CD4 T-cells (IFN-gamma) above 1/10,000 or IFN-gamma above 0.1 IU/ml (arbitrary value)
In group III, 3 of 4 patients with-initially-smear positive TB had percentages of CD27-neg. tuberculin-responsive CD4 T-cells above 50%, still one year after therapy, indicating that CD27 expression was not restored quickly after treatment (see
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).
Our results have demonstrated that CD27-expression on ex-vivo short term stimulated tuberculin specific CD4 T-cells is a highly discriminating biomarker for active pulmonary TB. It will also have utility for tracing TB contacts with latent TB infection. In this cohort of patients and controls the phenotype of tuberculin-reactive T-cells was more informative than quantitative measures of reactivity or target protein specificity.
While paradigms for compartmentalizing antigen dependent T-cell differentiation based on surface markers and functionality have been an area of major interest for a number of years
The degree of T-cell differentiation will likely depend on the circumstances, length, and frequency of exposure, and also the efficiency of the T-cell response in containing infection. These are obviously interdependent factors. In the 6 BCG vaccinated unexposed controls shown in
The fact that we did not observe more CD27-neg. tuberculin-reactive CD4 T-cells in patients with smear and culture negative TB than in control patients is of interest, since there was a clear distinction between unexposed donors and individuals with latent TB (
Recently it was shown in a TB mouse model that CD27-neg. lymphocytes isolated from lung produced more IFN-γ following stimulation with tuberculin than their CD27-pos. counterparts, which agrees with our findings in humans
The combination of low CCR7 but high CD28 expression that we observed on all tuberculin-reactive CD4 T-cells was recently associated with lung homing, which is indeed a prominent feature of tuberculin responsive CD4 T-cells
The finding that ESAT-6 reactivity was not a good marker for the diagnosis of active TB is in agreement with previous reports on acute TB patients and controls for example in Brazil and The Gambia
In order to rule out any bias related to subjective interpretation of flow-cytometry data the second part of the study was performed in a blinded fashion. This has fully confirmed our initial findings and demonstrated that gating of CD27-negative T-cells is a robust procedure. The fact that in some patients less than 50 IFN-γ producing cells were available for assessing the percentages of CD27-pos. and CD27-neg. cells reflects that our samples were stretched somewhat to accommodate several stimuli and antibody panels per sample in this pilot study. If only tuberculin had been used as a stimulus in combination with just one antibody panel, we would have had largely sufficient numbers of events in all TB patients.
In conclusion, our results argue that the phenotype of TB specific T-cells measured at the single cell level has considerable diagnostic potential and is more relevant than response quantity. A similar change of paradigm has already taken place in the HIV field
Since results can be obtained in less than 24 hours, this method could have utility in situations where diagnostic decisions are a matter of urgency. Smear and/or culture positive TB is a hazard to society and unnecessary hospitalizations are costly. It is, therefore, important to identify these patients rapidly. In this respect, our new assay is a considerable advance and calls for more research into using T-cell differentiation in diagnostic procedures in TB and other fields.
We would like to thank Ms. Anna Christina Nicholson, MA, for English corrections and Dr. Melanie Newport for helpful discussions.