Clinical immunology
Flow cytometric characteristics of alveolar lymphocytes (AL) obtained from the control group – proposal of normal value range of AL subsets in nonsmokers

Background: Immunological and cytological examination of bronchoalveolar lavage (BAL) fluid is frequently used for diagnostics and research of interstitial lung diseases (ILD). Flow cytometry (FC) is a common tool applied to investigations on biology of alveolar lymphocytes (AL). However, there is no consensus on the processing of immunophenotyping of BAL samples. As a consequence, the normal values of AL subsets are not definitely established.
The aim of the study was to determine the normal phenotype values of AL, using precise inclusion criteria in a relatively large cohort of healthy individuals. The data generated in this way could serve as a reference point for investigations and diagnostics of lower airways.
Material and methods: AL were obtained from BAL performed in 41 individuals free of any lung pathology, incl. 27 nonsmokers, NS, and 14 smokers, S. Direct double- and three-colour immunophenotyping was used for analysis of AL subsets, as well as for the markers of their activation and apoptosis susceptibility. Precise criteria of flow cytometry acquisition and analysis were employed. Mean fluorescence intensity (MFI) was determined for major AL subsets. The parallel typing of peripheral blood lymphocytes (PBL) was carried out.
Results: Both in NS and S, alveolar lymphocytes were almost exclusively T cells (90.1±1.4% and 89.3±1.6% respectively, median ± SEM). B and NK cells in BAL fluid occurred infrequently (2.7±0.4 and 4.2±0.8% in NS, 2.6±0.9 and 3.6±0.9% in S, respectively), as compared to peripheral lymphocytes (10.1±1.2% and 9.3±1.7% in NS, 17.9±3.4% and 14.0±1.9% in S, respectively, p<0.0001 for both). The AL were mostly CD45R0+ and CD95+ cells, regardless of analyzed subset (CD4 or CD8) and smoking status. Low BAL CD4/CD8 ratio in smokers, due to increased percentage of CD8+lymphocytes presenting T cytotoxic cell phenotype (CD8+CD11b–) was found (39.9±4.4% vs. 21.8±2.7% in NS, p<0.01). Only some AL CD8+ expressed suppressor cell phenotype (CD8+CD11b+), in opposite to the results obtained from peripheral blood. BAL fluid recovery (high value reflects increased content of alveolar cells vs. bronchial contamination) in NS (but not in S) was positively correlated with percentage of AL CD4+ (R Spearman +0.46, p=0.04) and CD4/CD8 ratio (R Spearman +0.45, p=0.02). MFI of CD3 and CD4 superficial antigens was significantly decreased on AL as compared to PBL (p<0.001 and p<0.01 resp.).
Conclusions: The normal value range was proposed for BAL lymphocyte immunophenotyping. The results support the hypothesis that T cells as a major subset of AL, they are chronically stimulated in lungs by specific antigens and carry the phenotype of primed memory/effector cells. Helper T cells are the lymphocytes that accumulate preferentially in alveoli in physiological conditions.