Stored Red Blood Cell Transfusion Induces Regulatory T Cells

Background

Allogeneic blood transfusion mediates immunosuppression in transfused recipients by an unknown mechanism. Regulatory T cells (T_regs) are suppressive CD4⁺CD25⁺Foxp3⁺ cells with a central role in immunosuppression in trauma victims, cancer patients, and transplant recipients. We hypothesized that transfusion-related immunosuppression is, in part, mediated by induction of T_regs, and this induction is attenuated with prestorage leukoreduction and accentuated with prolonged storage.

Study Design

Packed red blood cell (PRBC) units were obtained and 50% of PRBCs were leukoreduced (LR) before routine storage for 1 day or 42 days and the supernatant was collected. Normal human peripheral blood mononuclear cells (PBMCs) were exposed to 1-day NLR, 42-day NLR, 1-day LR, or 42-day LR PRBC supernatants or to PRBC storage solution or washed PRBC supernatant ± anti-CD3 stimulation, and analyzed by flow cytometry for Foxp3⁺ T_regs or CD25⁺-activated T cells. PRBC supernatants and cell culture supernatants were analyzed by immunoassay for interleukin (IL)-1β, IL-2, IL-4, IL-10, interferon-γ, tumor necrosis factor−α, and transforming growth factor−β. T_reg activity was evaluated by suppression assay.

Results

All PRBC groups induced T_regs compared with control media in anti−CD3-stimulated PBMCs, without alteration by LR or prolonged storage. PRBC supernatant did not alter nonspecific T-cell activation from control media. PRBC-induced T_regs were suppressive, inhibiting proliferation of T-responder cells. All cytokines measured decreased with storage in LR PRBC units and no cytokines were substantially elevated in cell supernatants exposed to PRBC supernatant. PRBC storage solution did not reproduce the effects of PRBC supernatant, and washed PRBC supernatant attenuated T_reg induction.

Conclusions

PRBC supernatant induces T_regs, but this induction is not altered by LR or prolonged storage. This induction appears to be independent of cytokines and is attenuated with washed PRBCs, implicating the plasma fraction.

Abbreviations and Acronyms: ABT, allogeneic blood transfusion, CFSE, carboxyfluorescein diacetate succinimidyl ester, Ctl, control media, IL, interleukin, LR, leukoreduced, NLR, nonleukoreduced, PBMC, peripheral blood mononuclear cell, PRBC, packed red blood cell, T_reg, regulatory T cell