1Universitätsmedizin Greifswald Klinik und Poliklinik für Innere Medizin B Greifswald, Deutschland
The phenotype of dilated cardiomyopathy (DCM) is characterized by the dilation of the left or both ventricles and a reduced systolic ejection fraction. The pathogenesis of DCM is complex, but an imbalance of humoral immunity and cardiac specific autoantibodies are associated with the development of DCM. B cell activating factor belonging to TNF family (BAFF) is primarily expressed by monocytes and controls the maturation of B cells. A pathophysiological high concentration of BAFF promotes the survival of autoantigen-reactive B cells, as known from studies concerning other autoimmune diseases where patients showed elevated serum levels of BAFF. In preliminary studies, we also observed significantly elevated BAFF levels in DCM patients.
In order to understand the role of BAFF in the pathogenesis of DCM better, we established a protocol to sequentially isolate and cultivate B cells, monocytes and T cells from blood samples of DCM patients and healthy controls for further stimulation experiments.
Blood samples were collected in CPTTM tubes and peripheral blood mononuclear cells (PBMCs) were isolated by centrifugation. We chose a positive separation approach applying magnetic labelled microbeads to separate CD20+ B cells from PBMCs, followed by monocyte and T cell separation simultaneously. CD14+ cell surface marker was used for monocytes and CD3+ marker for T cells. Flow cytometer analysis were performed for calculating cell counts, viability and purity. The freshly isolated cells were cultured up to 72 h for stimulation experiments with BAFF. During cultivation cell counts, purity and viability were monitored by flow cytometer measurements as well.
Viability and purity after isolation were higher than 95% for monocytes and T cells and higher than 75% for B cells. The greatest recovery rate was reached for monocytes (>90%), but B cell recovery (>80%) was also high. Although the recovery of T cells was lower (>50%), the total amount of T cells was high. During cultivation for 72 h there was a small decrease in viability of B cells, monocytes and T cells (>70%). The purity of B cells and monocytes remained greater than 80% during cultivation. T cells´ purity was constant at 99% over time.
We successfully established a protocol for the sequential isolation of human B cells, T cells and monocytes and their further maintenance under cell culture conditions for up to 72 h. This protocol will be used to collect blood samples from DCM patients and healthy controls. Furthermore, it will be applied for different stimulation approaches comparing gene expression and secretome profiles between blood cell subpopulations of DCM patients and healthy controls.