Mtb-specific CD27low CD4 T cells as markers of lung tissue destruction during pulmonary tuberculosis in humans.

PLoS One. 2012;7(8):e43733.

Nikitina IY, Kondratuk NA, Kosmiadi GA, Amansahedov RB, Vasilyeva IA, Ganusov VV, Lyadova IV.

Department of Immunology, Central Tuberculosis Research Institute, Moscow, Russia.

 

Abstract

BACKGROUND: Effector CD4 T cells represent a key component of the host’s anti-tuberculosis immune defense. Successful differentiation and functioning of effector lymphocytes protects the host against severe M. tuberculosis (Mtb) infection. On the other hand, effector T cell differentiation depends on disease severity/activity, as T cell responses are driven by antigenic and inflammatory stimuli released during infection. Thus, tuberculosis (TB) progression and the degree of effector CD4 T cell differentiation are interrelated, but the relationships are complex and not well understood. We have analyzed an association between the degree of Mtb-specific CD4 T cell differentiation and severity/activity of pulmonary TB infection.

METHODOLOGY/PRINCIPAL FINDINGS: The degree of CD4 T cell differentiation was assessed by measuring the percentages of highly differentiated CD27(low) cells within a population of Mtb- specific CD4 T lymphocytes (“CD27(low)IFN-γ(+)” cells). The percentages of CD27(low)IFN-γ+ cells were low in healthy donors (median, 33.1%) and TB contacts (21.8%) but increased in TB patients (47.3%, p<0.0005). Within the group of patients, the percentages of CD27(low)IFN-γ(+) cells were uniformly high in the lungs (>76%), but varied in blood (12-92%). The major correlate for the accumulation of CD27(low)IFN-γ(+) cells in blood was lung destruction (r = 0.65, p = 2.7 × 10(-7)). A cutoff of 47% of CD27(low)IFN-γ(+) cells discriminated patients with high and low degree of lung destruction (sensitivity 89%, specificity 74%); a decline in CD27(low)IFN-γ(+)cells following TB therapy correlated with repair and/or reduction of lung destruction (p<0.01).

CONCLUSIONS: Highly differentiated CD27(low) Mtb-specific (CD27(low)IFN-γ(+)) CD4 T cells accumulate in the lungs and circulate in the blood of patients with active pulmonary TB. Accumulation of CD27(low)IFN-γ(+) cells in the blood is associated with lung destruction. The findings indicate that there is no deficiency in CD4 T cell differentiation during TB; evaluation of CD27(low)IFN-γ(+) cells provides a valuable means to assess TB activity, lung destruction, and tissue repair following TB therapy.

PMID: 22937086

 

Summary

Due to a wide spread of M. tuberculosis (Mtb) infection, diagnostics of pulmonary tuberculosis (TB) represents an important health problem. Current methods for TB diagnostics, including microbiological, radiological and clinical examinations, have several limitations, especially in cases when mycobacteria cannot be identified in the sputum. Thus, biomarkers that can differentiate active TB (ATB) and latent TB infection (LTBI) and monitor TB treatment are needed for efficient disease diagnostics and control. Immunological methods have high potential for TB diagnostics due to high antigenic specificity of the immune response. This study has focused on the development of T-cell based immunological approach to evaluate TB activity and treatment efficacy.

We have investigated the relationships between activity of Mtb infection, severity of pulmonary TB and the degree of differentiation of Mtb-specific CD4 T cells. The degree of T cell differentiation was measured by determining the percentages of highly differentiated (CD27low) cells within a population of Mtb-specific CD4 T lymphocytes. The later were identified as CD4 T cells responding to Mtb antigens by producing IFN-g (‘‘CD27lowIFN-g+’’ cells). The percentages of CD27lowIFN-g+ cells were low in healthy donors (median, 33.1%) and TB contacts (21.8%) but increased in TB patients (47.3%, p<0.001). A cutoff of 35.1% distinguished TB patients from healthy donors and TB contacts with sensitivity 74% and specificity 83%. Within the group of TB patients, the percentages of CD27lowIFN-g+ cells were uniformly high in the lungs (76%), but varied in blood (12–92%). The major correlate for the accumulation of CD27lowIFN-g+in blood was lung destruction (r = 0.65, p = 2.7×10-7). A cutoff of 47% of CD27lowIFN-g+ cells discriminated TB patients with high and low degree of lung destruction (sensitivity 89%, specificity 74%); a decline in CD27lowIFN-g+ cells following TB therapy correlated with repair and/or reduction of lung destruction (p<0.01).

These findings signify that determination of the percentages of CD27lowIFN-g+ cells allows evaluating activity of Mtb infection, severity of Mtb-induced lung tissue destruction and its repair following TB treatment (process that until now have been monitored only by X-ray examination).

 

Supplement

TB is a global challenge. It is estimated that approximately one third of the human population are infected with Mtb and more that 8 million cases occurs annually. In patients with less-extensive pulmonary TB, extrapulmonary TB and in children, microbiological (sputum smear, culture) and molecular biological (PCR) diagnosis is often limited due to the lack of identifiable Mtb in the sputum. Thus, biomarkers to diagnose TB, differentiate between ATB and LTBI and monitor TB treatment are needed.

Effector CD4 T cells represent a key component of the host’s anti-tuberculosis immune defense. Following the infection, effector CD4 T cells differentiate from naïve lymphocytes. Our recent studies in mice have demonstrated that Mtb infection induces the differentiation of CD4 T cells into a highly-differentiated subset, expressing CD27low phenotype and that this differentiation occurs directly in Mtb-infected lung tissue. We hypothesized that the degree of CD4 T cell differentiation (evaluated as a percent of CD27low cells within the whole population of Mtb-specific CD4 T cells) may be used to measure TB activity.

We therefore compared the percentages of CD27low cells within a population of Mtb-specific CD4 T cells in healthy individuals and TB patients. The study included 50 patients with ATB, 21 healthy hospital employees working in close contact with TB patients (“contacts”) and 15 healthy donors without a known contact with Mtb infection.

Comparing the percentages of CD27lowIFN-g+ cells in the blood of TB patients, contacts and healthy donors revealed that the percentages were low in healthy donors and contacts (median, 33.1% and 21.8%, respectively) and significantly higher in TB patients (47.3%, p<0.001). A cutoff of 35.1% distinguished TB patients from healthy donors and TB contacts with sensitivity 74% and specificity 83%; a cut-off of 31.2% discriminated TB patients and contacts with specificity 91% and sensitivity 82%. Thus, determining the percentages of CD27lowIFN-g+ cells in blood can be used for discriminate ATB from LTBI.

Next, we compared the percentages of CD27lowIFN-g+ cells in the blood and in the lungs  of TB patients. The percentages were high in the lungs (>76%) and varied in blood (12–92%). The major factors that explained the variation in CD27lowIFN-g+ cells in the blood were the degree of lung destruction and clinical disease severity (r= 0.65, p = 2.7×10-7; r=0.63, p=7.7×10-7).

Because the degree of lung tissue destruction is an important pathogenic factor that causes TB progression, morbidity, and bacillary spread, we asked whether evaluation of CD27lowIFN-g+ cells could be used as a means to estimate pulmonary destruction. We found that a cutoff of 47% of CD27lowIFN-g+ cells allowed distinguishing patients with high and low degree of lung destruction (sensitivity 89%, specificity 74%). Examples of X-ray computer tomography with different degrees of tissue destruction are shown in Figure 1 A-D.

Finally, we evaluated whether the percentages of CD27lowIFN-g+ cells changed following the treatment and could be used to monitor treatment efficacy. Totally, 22 patients with initially elevated (>35.1%) percentages of CD27lowIFN-g+ cells were included in the analysis. The percentages of CD27lowIFN-g+ cells and the degree of lung tissue destruction were evaluated at the beginning of treatment and 2 months later (Figure 1 E-J). The results showed that a decline in the percentages of CD27lowIFN-g+ cells was indicative of the repair of lung tissue destruction (determined based on X-ray/computer tomography data).

The importance of this study: These findings signify that determining the percentages of CD27lowIFN-g+ cells in the blood allows evaluating activity of Mtb infection, severity of Mtb-induced lung tissue destruction, and its repair following TB treatment (processes that until now have been monitored only by X-ray examination).

Irina Yu. Nikitina-jpg

Figure 1. Percentages of CD27lowIFN-γ+ cells mirror the degree of lung tissue destruction.

A-D, Examples of computer tomography and corresponding percentages of CD27lowIFN-γ+ cells in the blood of TB patients.

A, score 0, no destruction; B, score 1, one small (<2 cm) destruction; C, score 2, one large (³2 cm) destruction; D, score 3, multiple lung destructions. Arrows, lung destructions. Asterisk, infiltrative focus without destruction.

E-F, Following anti-TB therapy, percentages of CD27lowIFN-γ+ cells decline parallel to reduction/repair of lung destruction. The percentages of CD27lowIFN-γ+ cells were determined at the beginning of treatment and 2 months later. Depending on the results, all patients were divided into three groups (E-G):

E, “CD27lowIFN-γ+ – high” group, Percentages of CD27lowIFN-γ+ cells did not decline following anti-TB therapy;

F, “CD27lowIFN-γ+ – reduced” group, Percentages of CD27lowIFN-γ+ cells declined to become below the 47% threshold, but remained above 35.1% threshold (upper limit of norm);

G, “CD27lowIFN-γ+ – normalized” group, Percentages of CD27lowIFN-γ+ cells declined to become below the 35.1% threshold.

Dotted lines show 35.1 and 47% thresholds.

H-J, Diagrams showing changes in lung destruction for each group of TB patients following 2-mo therapy.

H, Changes in lung destruction in “CD27lowIFN-γ+ – high” group of TB patients; I, Changes in lung destruction in “CD27lowIFN-γ+ – reduced” group of TB patients; J, Changes in lung destruction in “CD27lowIFN-γ+ – normalized” group of TB patients. Closed segments, no improvement; squared segments, reduction of lung destruction; open segments, normalization (repair of lung destruction).

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