Introduction: PAP is due to accumulation of phospholipids and lipoproteins in the alveoli. WLL is still the treatment of choice. Light microscopy examination of WLL fluid reveals a moderate to high lymphocytosis but lymphocyte subpopulations have not been further characterized at yet.
Aims: To investigate T lymphocyte subsets in WLL fluid from patients with PAP.
Methods: From the first recovered fluid portion during WLL, samples of 50 mL each were collected and analyzed fresh. Pancoll density-gradient centrifugation was performed to separate mononuclear (MN) cells. MACS^® enrichment was used to separate T lymphocytes from remaining MN cells and acellular particles after adding anti-CD3 microbeads to the MN cell layer. Flow cytometry for T cell immunophenotyping was performed by using a 9-color antibody panel and a FACSAria^? Fusion cytometer (Becton Dickinson, Germany). Zombie UV^? fluorescent dye was used to assess cell viability. T lymphocyte counts were expressed as proportion of CD3⁺ viable lymphocyte counts.
Results: WLL fluid from 3 consecutive PAP patients (2 M, 1 F, age 44±12) who underwent therapeutic WLL was analyzed. The mean T lymphocyte count was 87±1%. CD4⁺ T cells were the dominant T cell subset across all samples (73±7%), followed by CD8⁺ cytotoxic T cells (19±7%). Th17.1 cells were the most frequent Th cell subset (33±14%), followed by Th1 cells (29±2%), Tregs (17±6%), Th17 cells (4±2%), and Th2 cells (0.7±0.2%).
Conclusions: Our study identified different T lymphocyte subsets, mostly related to autoimmunity, in WLL fluid obtained from PAP patients. Flow cytometry may be used to unravel pathogenetic mechanisms of this rare disease.