T-cell activation profiles in different granulomatous interstitial lung diseases--a role for CD8+CD28(null) cells?

Abstract

Lymphocytes play a crucial role in lung inflammation. Different interstitial lung diseases may show distinct lymphocyte activation profiles. The aim of this study was to examine the expression of a variety of activation markers on T lymphocyte subsets from blood and bronchoalveolar lavage fluid (BALF) of patients with different granulomatous interstitial lung diseases and healthy controls. Bronchoalveolar lavage cells and blood cells from 23 sarcoidosis patients, seven patients with hypersensitivity pneumonitis and 24 healthy controls were analysed. Lymphocyte activation status was determined by flow cytometry. Lymphocytes were stained with antibodies against CD3, CD4, CD8, CD25, CD28, CD69, very late antigen-1 (VLA)-1, VLA-4 and human leucocyte antigen D-related (HLA-DR). In general, CD28, CD69 and VLA-1 expression on BALF CD4+ lymphocytes and HLA-DR expression on BALF CD8+ lymphocytes was different in patients with hypersensitivity pneumonitis and sarcoidosis patients with parenchymal involvement. This BALF lymphocyte phenotype correlated with carbon monoxide diffusing lung capacity (Dlco) values across interstitial lung diseases (ILD) (r2 = 0.48, P = 0.0002). In sarcoidosis patients, CD8+CD28(null) blood lymphocytes correlated with lower Dlco values (r = -0.66, P = 0.004), chronic BALF lymphocyte activation phenotype (r2 = 0.65, P < 0.0001), radiographic staging (stage I versus stage II and higher, P = 0.006) and with the need for corticosteroid treatment (P = 0.001). Higher expression of CD69, VLA-1 and HLA-DR and lower expression of CD28 on BALF lymphocytes suggests prolonged stimulation and chronic lymphocyte activation in patients with ILD. In sarcoidosis, blood CD8+CD28(null) cells might be a new biomarker for disease severity but needs further investigation.

Fig. 1

Gating strategy. A representative side-scatter versus forward-scatter plot and lymphocyte gate is shown in peripheral blood (PB) and bronchoalveolar lavage fluid (BALF) samples. Expression of activation markers was analysed on CD4⁺ and CD8⁺ cells within the lymphocyte gate (a,b,c). Back gating of gated cell populations double-stained for T cell receptor (TCR) marker CD3 and an activation marker into the side scatter–forward scatter (SSC–FSC) plot showed that the gated cells fell within the SSC–FSC lymphocyte gate, hence no cells were omitted using the original lymphocyte gate (d,e).

Fig. 2

Fluorescence histograms presenting CD28, CD69 and very late antigen (VLA)-1 expression on bronchoalveolar lavage fluid CD4⁺ cells and human leucocyte antigen D-related (HLA-DR) expression on BALF CD8⁺ cells from one representative healthy control (HC), sarcoidosis (SARC) stage I, SARC stage IV and hypersensitivity pneumonitis (HP) subject.

Fig. 3

Scatter diagram illustrating the sum of ranks of CD25, CD28, CD69, very late antigen (VLA)-1 and human leucocyte antigen D-related (HLA-DR) expression on bronchoalveolar lavage fluid (BALF) CD4⁺ lymphocytes and BALF CD8⁺ lymphocytes in sarcoidosis patients without parenchymal involvement (radiographic stage I), sarcoidosis patients with parenchymal involvement (radiographic stage II and higher) and patients with hypersensitivity pneumonitis (HP). VLA-4 expression was 100% in both BALF lymphocyte subsets in all patients, hence VLA-4 expression was excluded from sum of rank analysis (all subjects would have been assigned the same rank number, i.e. 1).

Fig. 7

Scatter plot of the percentage of peripheral blood CD8⁺CD28^null T cells in sarcoidosis patients (n = 21) at presentation and the need for corticosteroid treatment within 2 years after presentation. The dotted line indicates 50% level. Ten patients had CD8⁺CD28^null values above 50%. Of these patients, seven needed medication. None of the patients with CD8⁺CD28^null values lower than 50% received corticosteroids within 2 years after presentation. Fisher's exact test: P = 0·001.

Fig. 6

Scatter diagram illustrating the correlation between the percentage of peripheral blood (PB) CD8⁺CD28^null T cells and the sum of ranks of the individual significant correlations with the percentage of bronchoalveolar lavage fluid (BALF) CD4⁺CD28⁺, CD4⁺ very late antigen (VLA-1)⁺, CD8⁺CD25⁺, CD8⁺VLA-1⁺ and CD8⁺ human leucocyte antigen D-related (HLA-DR)⁺ lymphocytes from sarcoidosis patients.

Fig. 5

Scatter diagram illustrating the correlation between the percentage of peripheral blood (PB) CD8⁺CD28^null T cells and carbon monoxide diffusing lung capacity (Dlco) in 17 sarcoidosis patients. Inlay presents the percentage of PB CD8⁺CD28^null T cells between sarcoidosis patients without parenchymal involvement, radiographic stage I (n = 14) and with parenchymal involvement, radiographic stage II and higher (n = 9) at presentation.

Fig. 4

Fluorescence histograms showing blood CD8⁺CD28^null and CD8⁺CD28⁺ cell populations in sarcoidosis patients (upper row) and healthy controls (lower row).