Loss of Receptor on Tuberculin-Reactive T-Cells Marks Active Pulmonary Tuberculosis

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 naive/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

I am not the one I used to be : A qualitative study about experiences of identity development among unaccompanied boys from Afghanistan.

The purpose of the study is to understand/investigate how unaccompanied boys from Afghanistan experience their identity and identity development in interaction with Swedish culture and Swedish society. Qualitative data collection methods have been used in the form of semi-structured interviews. The material has been analyzed on the basis of identity development theory and the theory of ethnic identity. Based on the study's gathered empirical data and analysis, important results found that experiences of identity development in unaccompanied boys are strongly influenced by Swedish society and Swedish culture. The majority of the informants expressed that their identity has been affected and changed a lot during the time they lived in Sweden and that influence has helped these young people to develop a new identity. In order to be a part of the majority society and integrate well into society, this influence on identity has been positive and important for the informants. The study’s results also show that the informants have managed to find a balance between the new identity and their ethnic identity

Responder frequencies^a in different TB related tests in the blind study.

a<p>not all test were performed in all individuals, numbers refer to positive test results per tested individuals</p>b<p>frequency of tuberculin-reactive CD4 T-cells (IFN-gamma) above 1/10,000 or IFN-gamma above 0.1 IU/ml (arbitrary value)</p>c<p>frequency of ESAT-6 reactive CD4 T-cells (IFN-gamma) above 1/10,000 or IFN-gamma above 0.1 IU/ml (arbitrary value)</p

Crosstabulation of results in blind study.

<p>Crosstabulation of results in blind study.</p

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

<p>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.</p

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

<p>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.</p

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

<p>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).</p

Tuberculin-Specific T Cells Are Reduced in Active Pulmonary Tuberculosis Compared to LTBI or Status Post BCG Vaccination

Functional characteristics of tuberculosis (TB)–specific CD4 T cells were studied in clinically active pulmonary TB (n = 21) and high TB exposure including LTBI (n = 17). Following tuberculin stimulation, activated CD4 T cells were identified by flow-cytometry (CD154 up-regulation, degranulation, interferon γ [IFN-γ], tumor necrosis factor α [TNF-α], and interleukin 2 [IL-2\ production). Interestingly, CD154 up-regulation accounted for ∼80% of activated CD4 T cells in the active TB group but just 40% in the controls, whereas IFN-γ accounted for only ∼50% of activated cells in each group. The frequencies of CD4 T cells displaying at least 1 activation marker discriminated better between the groups than those displaying degranulation or IFN-γ production alone

Patients included in the blind study.

a<p>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</p>b<p>results exceeding 1 positive event per 10,000 cells were counted as positive and are shown in bold</p>c<p>some of these results exceed the threshold for positivity published for a commercial assay (Quantiferon Gold, Cellestis) of 0.35 IU.</p