How and when to pick up the best signals from markers associated with T-regulatory cells?

Regulatory T (Treg) cells are important tools with the purpose to control and regulate the immune system. These cells use FOXP3, TGF-beta, CTLA-4 and sCTLA-4 to regulate other T-cells. Since cryopreservation of peripheral blood mononuclear cells (PBMC) is a convenient way to handle samples, we investigated whether these cells will change their mRNA expression of Treg associated markers, as well as secretion of TGF-beta1, after cryopreservation. Additionally, we aimed to investigate the mRNA expression after two different time intervals of in vitro antigen stimulation. PBMC from healthy adults were stimulated either fresh (48/96 h) or after cryopreservation (48 h), with PHA, tetanus toxoid, beta-lactoglobulin, ovalbumin or in culture medium exclusively (spontaneous). The mRNA expression of FOXP3, TGF-beta, CTLA-4 and sCTLA-4 were studied with multiplex real-time RT-PCR and TGF-beta1 with ELISA. Cryopreserved PBMC were appropriate for detection of the Treg associated markers FOXP3, TGF-beta, CTLA-4 and sCTLA-4 expressed spontaneously. Also antigen-induced mRNA expression of CTLA-4, sCTLA-4 and TGF-beta and secreted TGF-beta1, with the exception of FOXP3, preserved a stable transcription activity after cryopreservation. Further, a stimulation period of 48 h in general revealed the highest mRNA expression of the markers studied. In conclusion, cryopreserved cells are in general suitable for studying Treg associated markers.