Packed red blood cells suppress T-cell proliferation through a process involving cell-cell contact

Background:: Packed red blood cell (PRBC) transfusion suppresses immunity and increases morbidity and mortality. Leukocyte reduction has failed to abrogate these effects, thus implicating red blood cells themselves or their components. PRBC impair proliferation of immortal (Jurkat) T cells by depleting arginine from the extracellular environment. The effect of PRBC on isolated ex vivo T-cell proliferation has not been reported. We hypothesize that PRBCs depress mitogen-stimulated proliferation in isolated human and mouse T cells. Methods:: Human peripheral T cells were isolated by Ficoll-Hypaque gradient, purified by magnetic separation, and stimulated with anti-CD3 or anti-CD28. DO11.10 transgenic mouse splenic T cells were stimulated with ovalbumin. Cells were cultured at 1 × 10⁶/mL in 96-well plates or in 24-transwell plates in the presence of PRBC (0.015-5% by volume, stored for 4-6 weeks). In culture media, arginine and citrulline were varied. Proliferation was measured at 72 hours by thymidine incorporation. T-cell viability, apoptosis, and receptor ζ chain were measured by flow cytometry. Results:: PRBC significantly depressed human peripheral and mouse splenic T-cell proliferation in a dose-dependent manner. PRBC arginase blockade by N-omega-hydroxy-nor-l- arginine only partly restored proliferation. Cell contact was required in both cell types for maximal effect. Depressed ζ chain in human peripheral T cells was partly restored by arginase blockade. Salvage by high-dose arginine and citrulline was unsuccessful. Decreased proliferation was not related to cell death. Conclusion:: PRBC suppresses mitogen-stimulated human and antigen-stimulated mouse T-cell proliferation by mechanisms independent of arginine depletion. This is a novel mechanism for transfusion-associated immune suppression.