T cell analyses in blood, BAL and induced sputum from lung transplant recipients

1. Sputum BALF 21% Left panel: whole blood, T cells gated by morphology and expression of CD3 (APC). Histogram shows expression of CCR7 (FITC) Middle panel: BALF, T cells gated by expression of CD45 (PE-Cy7) and CD3 (APC). Histogram shows expression of CCR7 (FITC) and CD25 (APC-Cy7) Right panel: Induced sputum, T cells gated by expression of CD45 (PE-Cy7) and CD3 (APC). Histogram shows expression of CCR7 (FITC) and CD25 (APC-Cy7) T cell analyses in blood, BAL and induced sputum from lung transplant recipients Are Martin Holm MD PhD (1,2) Liv Ingunn Bjoner Sikkeland MSc PhD (1,2) Tonje Bøyum Riste MSc (1,2) May Brit Lund MD PhD (1) Øystein Bjørtuft MD PhD (1) Johny Kongerud MD PhD (1,2) Neil Alexis PhD (3) (1) Department of Respiratory Medicine, Rikshopitalet, Oslo University Hospital, Oslo, Norway (2) Medical Faculty, University of Oslo, Oslo, Norway (3) Department of Pediatrics, Division of Immunology and Infectious Disease, University of North Carolina School of Medicine, Chapel Hill, NC Corresponding author: Are M. Holm, a.m.holm@medisin.uio.no The authors have no conflicts of interest do disclose • BACKGROUND • Bronchiolitis obliterans syndrome (BOS) is the most important limitation to long term survival after lung transplantation. The mechanisms are unknown, and no reliable biomarkers have been found. • We hypothesized that analyzing T cells from the less invasive method of induced sputum (IS) is an equally informative method as bronchoalveolar lavage fluid (BALF) to obtain possible biomarkers in the surveillance of lung transplant recipients. • METHODS • Double lung transplant recipients having either stable lung function (FEV1>80% of max. values since transplant) or BOS were included in either a longitudinal study with repeated sampling (1, 3, 6 months and 1 year post transplant) or in a cross-sectional study (at 1 to 5 years post transplant). Cells were isolated from blood, IS and BALF and analyzed by flow cytometry. • No serious adverse events occurred during BAL or induced sputum procedures Cytospin slides from BALF (left) and induced sputum (right) Upper left panel shows percentage of CD3+ cells (T cells) related to CD45+ cells (all leukocytes) in blood, BALF and induced sputum as determined by flow cytometry. Upper right panel shows the ratio of CD4+ to CD8+ among these T cells, lower left panel shows the percentage of T cells that are CCR7+ and lower right panel shows the percentage of T cells that are CD25+. • RESULTS • T cells were available for analyses in 25/31 IS samples and 17/22 BALF samples. • IS had lower overall % of T cells vs BALF and blood. Several markers associated with BOS were detectable in IS: CD4/CD8 T cell ratios were similar in peripheral blood, BALF and IS, but T cells in BALF and IS were predominantly CCR7- (effector memory type) and IS had elevated CD25 vs BALF and blood. CONCLUSION Induced sputum is a safe and feasible method for analyzing T cells from lung transplant recipients. Further studies using induced sputum may reveal useful biomarkers for BOS.