Packed red blood cells inhibit T-cell activation via ROS-dependent signaling pathways

Numerous observations indicate that red blood cells (RBCs) affect T-cell activation and proliferation. We have studied effects of packed RBCs (PRBCs) on T-cell-receptor (TCR) signaling and the molecular mechanisms whereby (P)RBCs modulate T-cell activation. In line with previous reports, PRBCs attenuated the expression of T-cell activation markers CD25 and CD69 upon co-stimulation via CD3/CD28. In addition, T-cell proliferation and cytokine expression were markedly reduced when T-cells were stimulated in presence of PRBCs. Inhibitory activity of PRBCs required direct cell-cell contact and intact PRBCs. The production of activation-induced cellular reactive oxygen species (ROS), which act as second messengers in T-cells, was completely abrogated to levels of unstimulated T-cells in presence of PRBCs. Phosphorylation of the TCR-related zeta-chain and thus proximal TCR signal transduction was unaffected by PRBCs, ruling out mechanisms based on secreted factors and steric interaction restrictions. Only downstream signaling events requiring ROS for full functionality were affected, confirmed by an untargeted mass spectrometry-based phosphoproteomics approach. PRBCs inhibited T-cell activation more efficiently than did treatment with 1 mM of the anti-oxidant N-acetyl cysteine. Taken together, our data suggest that inflammation-related radical reactions are modulated by PRBCs. These immunomodulating effects might be responsible for clinical observations associated with transfusion of PRBCs.