Increased cytotoxic T-cells in the airways of adults with former bronchopulmonary dysplasia

Abstract

Rationale: Bronchopulmonary dysplasia (BPD) in preterm-born infants is a risk factor for chronic airway obstruction in adulthood. Cytotoxic T-cells are implicated in COPD, but their involvement in BPD is not known.

Objectives: To characterise the distribution of airway T-cell subsets in adults with a history of BPD.

Methods: Young adults with former BPD (n=22; median age 19.6 years), age-matched adults born preterm (n=22), patients with allergic asthma born at term (n=22) and healthy control subjects born at term (n=24) underwent bronchoalveolar lavage (BAL). T-cell subsets in BAL were analysed using flow cytometry.

Results: The total number of cells and the differential cell counts in BAL were similar among the study groups. The percentage of CD3⁺CD8⁺ T-cells was higher (p=0.005) and the proportion of CD3⁺CD4⁺ T-cells was reduced (p=0.01) in the BPD group, resulting in a lower CD4/CD8 ratio (p=0.007) compared to the healthy controls (median 2.2 versus 5.3). In BPD and preterm-born study subjects, both CD3⁺CD4⁺ T-cells (r_s=0.38, p=0.03) and CD4/CD8 ratio (r_s=0.44, p=0.01) correlated positively with forced expiratory volume in 1 s (FEV₁). Furthermore, CD3⁺CD8⁺ T-cells were negatively correlated with both FEV₁ and FEV₁/forced vital capacity (r_s= -0.44, p=0.09 and r_s= -0.41, p=0.01, respectively).

Conclusions: Young adults with former BPD have a T-cell subset pattern in the airways resembling features of COPD. Our findings are compatible with the hypothesis that CD3⁺CD8⁺ T-cells are involved in mechanisms behind chronic airway obstruction in these patients.

FIGURE 1

Representative flow cytometry dot plots of a) CD8⁺ (y-axis) and CD4⁺ (x-axis) among CD3⁺ T-cells from bronchoalveolar lavage (BAL); b) T-cell differentiation among CD3⁺ T-cells from BAL; and c) B-cells among live cells from BAL. Samples were taken from three different study subjects. PE: phycoerythrin; PerCP: peridin chlorophyll protein; Cy: cyanine dye; APC: allophycocyanine.

FIGURE 2

Dot plots show the percentage in bronchoalveolar lavage CD3⁺ cells, with boxes demonstrating interquartile range, horizontal line indicating the median, and whiskers showing the 95% confidence interval. a) CD3⁺CD4⁺ T-cells (T-helper cells) (%); b) CD3⁺CD8⁺ T-cells (cytotoxic T-cells) (%); c) CD4/CD8 ratio of CD3⁺ cells; d) CD3⁺CD8⁺CD69⁺ (activated cytotoxic T-cells) (%). All cells were gated for live cells. Analysis with Mann–Whitney U-test. BPD: bronchopulmonary dysplasia.

FIGURE 3

Characterisation of T-cell differentiation in bronchoalveolar lavage CD3⁺ cells. The dot plots show the percentage, and the boxes demonstrate interquartile range with the horizontal line for median for a) CD27⁺CD45RA⁺ (naïve cells) (%); b) CD27⁺CD45RA⁻ (central memory cells) (%); c) CD27⁻CD45RA⁻ (effector memory cells) (%); and d) CD27⁻CD45RA⁺ (effector cells) (%) in healthy control, asthma, preterm and bronchopulmonary dysplasia (BPD) groups. Analysis with Mann–Whitney U-test.

FIGURE 4

Dot plot showing the percentage of FoxP3⁺ T-cells in bronchoalveolar lavage CD3⁺ cells, with boxes indicating interquartile range, horizontal lines indicating the median and whiskers showing the 95% confidence interval. a) CD3⁺CD4⁺FoxP3⁺ T-cells (%); b) CD3⁺CD8+FoxP3⁺ T-cells (%). Analysis with Mann–Whitney U-test. BPD: bronchopulmonary dysplasia.

FIGURE 5

Protein concentrations of a) granzyme B and b) perforin were measured by ELISA in concentrated bronchoalveolar lavage (BAL) fluid. Dot plots show the distribution of detected levels of granzyme B and perforin, with boxes demonstrating interquartile range, horizontal line showing the median, and whiskers showing the 95% confidence interval. The dotted line represents a) the lowest level of quantitation assay range for granzyme B and b) the limit of detection for perforin. Analysis with Mann–Whitney U-test. BPD: bronchopulmonary dysplasia.

FIGURE 6

Correlation in pooled preterm group (preterm+bronchopulmonary dysplasia (BPD)) between a) CD3⁺CD4⁺ T-cells (%) in bronchoalveolar lavage (BAL) and post-bronchodilator forced expiratory volume in 1 s (FEV₁) z-scores; b) CD3⁺CD8⁺ T-cells (%) in BAL and post-bronchodilator FEV₁ z-scores; c) CD4/CD8 ratio of CD3⁺ cells in BAL and post-bronchodilator FEV₁ z-scores. r_s: Spearman rank correlation coefficient.