Induced cytokine response of human PMBC-cultures: Correlation of gene expression and secretion profiling and the effect of cryopreservation

The immune system is regulated by the complex interaction of multiple cytokines, which are secreted signaling molecules affecting other cells. In this work, we studied the cytokine response to several well-known stimulants, such as OKT-3, Con A, PWM, and SEB. Healthy donor cells (PBMCs) were cultivated for up to 72 h and the mRNA levels and cytokine release of four key cytokines (IL-2, IL-4, IFN-γ, and TNF-α) were analyzed by RT-PCR and bead-based multiplex analyses. The generated cytokine profiles showed characteristic expression patterns and secretion kinetics for each cytokine and substance. PWM/SEB and OKT-3 led to a very fast and long-lasting immune response, whereas Con A induced the slowest cytokine production. Cytokine concentrations also differed greatly. The highest IFN-γ concentration was 1000 times higher than the respective IL-4 concentration. Gene expression and cytokine concentration profiles were strongly correlated during the time course.The chronological response of the donors’ cytokine profiles coincided, but showed individual characteristics regarding the strength of the cytokine release.The comparison of stimulation experiments using freshly isolated and cryopreserved PBMCs showed that, for the observation of an immunological response at early points in time, gene expression experiments are more reliable than the measurement of cytokines in the cell culture supernatant. However, the freezing of cells influences the response significantly. The measurement of secreted proteins is the superior method at later points in time.

► We compare immunologic response of cryopreserved and freshly isolated PBMC.
► We examine the gene expression and level of secretion of four key cytokines.
► Use of different stimulants leads to substance and donor specific cytokine profiles.
► Differences between secreted cytokine levels are negligible after 24 h.
► Gene expression rates and secretion levels correlate strongly in time and strength.