PIF direct immune regulation: Blocks mitogen-activated PBMCs proliferation, promotes TH2/TH1 bias, independent of Ca2+

PreImplantation Factor (PIF<inf>9&15</inf>) secreted by viable embryos exerts an essential transplant acceptance and immune regulatory role in pregnancy. Synthetic PIF replicates endogenous PIF's effect in pregnant and non-pregnant immune disorder models. PIF binds macrophages to regulate CD3/CD28-induced T-cell response. We present evidence that PIF regulates the co-stimulatory T-cell receptor, CD2, which binds to and is activated by phytohemagglutinin (PHA), a potent mitogen, confirming PIF's ability to systemically respond to diverse immune stimulants. PIF's effect on PHA-activated PBMC (male and non-pregnant females) proliferation and cytokine secretion was tested, showing that both PIF<inf>9&15</inf> block PHA-induced PBMC proliferation and promote anti-inflammatory IL10 secretion, while reducing pro-inflammatory IFNγ secretion. Thus favoring a T<inf>H</inf>2 cytokine bias. Surface plasmon resonance spectroscopy, immunocytochemistry and Flex station experiments reveal that PIF effect is direct. PIF targets intracellular targets but does not affect early Ca²⁺ mobilization. By promoting the CD2 receptor in activated T-cells and through inhibition of co-ligand CD58 expression, PIF regulates antigen-presenting cell (APC)-T-cell interactions required for PHA action. Structure-based design demonstrated that PIF<inf>15</inf> offers improved target specificity as compared to PIF<inf>9</inf>. Collectively, PIF directly regulates mitogen-induced PBMC activation. Results support PIF translation for therapy of immune disorders.