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tored Red Blood Cell Transfusionnduces Regulatory T Cellsoel M Baumgartner, MD, Christopher C Silliman, MD, PhD, Ernest E Moore, MD, FACS,nirban Banerjee, PhD, Martin D McCarter, MD, FACS

BACKGROUND: Allogeneic blood transfusion mediates immunosuppression in transfused recipients by an un-known mechanism. Regulatory T cells (Tregs) are suppressive CD4�CD25�Foxp3� cells witha central role in immunosuppression in trauma victims, cancer patients, and transplant recip-ients. We hypothesized that transfusion-related immunosuppression is, in part, mediated byinduction of Tregs, and this induction is attenuated with prestorage leukoreduction and accen-tuated 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 humanperipheral blood mononuclear cells (PBMCs) were exposed to 1-day NLR, 42-day NLR, 1-dayLR, or 42-day LR PRBC supernatants or to PRBC storage solution or washed PRBC superna-tant � anti-CD3 stimulation, and analyzed by flow cytometry for Foxp3� Tregs or CD25�-activated T cells. PRBC supernatants and cell culture supernatants were analyzed by immuno-assay for interleukin (IL)-1�, IL-2, IL-4, IL-10, interferon-�, tumor necrosis factor��, andtransforming growth factor��. Treg activity was evaluated by suppression assay.

RESULTS: All PRBC groups induced Tregs compared with control media in anti�CD3-stimulatedPBMCs, without alteration by LR or prolonged storage. PRBC supernatant did not alternonspecific T-cell activation from control media. PRBC-induced Tregs were suppressive, inhib-iting proliferation of T-responder cells. All cytokines measured decreased with storage in LRPRBC units and no cytokines were substantially elevated in cell supernatants exposed to PRBCsupernatant. PRBC storage solution did not reproduce the effects of PRBC supernatant, andwashed PRBC supernatant attenuated Treg induction.

CONCLUSIONS: PRBC supernatant induces Tregs, 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. (J Am Coll Surg 2009;208:110–119. © 2008 by the American
College of Surgeons)
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llogeneic blood transfusion (ABT) is recognized to haveiverse immunomodulatory effects. ABT is associated withproinflammatory response, as suggested by its mechanis-

ic role in multisystem organ failure and transfusion-relatedcute lung injury.1,2 ABT also has immunosuppressive ef-ects, which have been appreciated since it was first re-orted by Opelz and colleagues3 in 1973 that pretransplan-ation ABT could improve renal-allograft survival. Inddition, ABT has other immune regulatory effects be-

isclosure Information: Nothing to disclose.

eceived June 20, 2008; Revised August 16, 2008; Accepted August 18,008.rom the Departments of Surgery (Baumgartner, Moore, Banerjee,cCarter) and Pediatrics (Silliman), University of Colorado Denver, Den-

er, and Bonfils Blood Center, Denver, CO (Silliman).orrespondence address: Joel M Baumgartner, MD, Department of Surgery,

kniversity of Colorado Denver, 4200 E 9th Ave C-320, Denver, CO 80262.

1102008 by the American College of Surgeons

ublished by Elsevier Inc.

ause it is associated with increased cancer recurrence, de-reased severity of autoimmune disease, and increased in-ection risk.4-6

Storage of packed red blood cells (PRBCs) exaggerateshe immune modulation of ABT because it results in pro-ressive accumulation of immunologic mediators until theast day at which the PRBCs can be transfused (day 42).7-10

restorage leukoreduction (LR) has been proposed to ame-iorate immunosuppression from donor leukocytes oronor leukocyte-derived mediators. LR diminishes themount of transfused cytokines and other immunologicediators, and this can attenuate the immunosuppressive

ffects of ABT by reduction of functional defects in themmune response.11,12

A variety of mediators for transfusion-related immuno-uppression have been proposed, including allogeneic leu-
ocytes, leukocyte-derived soluble mediators, and soluble
ISSN 1072-7515/09/$36.00doi:10.1016/j.jamcollsurg.2008.08.012

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111Vol. 208, No. 1, January 2009 Baumgartner et al Stored RBC Transfusion Induces Regulatory T Cells

LA molecules, although the precise mechanisms ofransfusion-related immunosuppression remain uncer-ain.13 Given their broad repertoire of targets and theirritical importance in immune regulation, regulatory Tells (Tregs) represent a potential mechanism of transfusion-elated immunosuppression. This population of CD4�

D25� T cells promotes immune tolerance, prevents au-oimmunity, and is characterized by unique expression ofoxp3, a transcription factor of critical importance for Treg

evelopment and function in the forkhead/winged-helixamily of transcriptional regulators.14,15 Tregs suppressD4� and CD8� effector T-cell�mediated immunity,

nd inhibit the function of dendritic cells to substantiallylunt the immune response.16,17 Treg-mediated immuno-uppression has been implicated in the immunopathologyf trauma and sepsis, as injured patients contain elevatedevels of hypersuppressive circulating Tregs.

18,19 Thesemmune-suppressive cells also infiltrate tumors and canause subversion of the antitumor immune response, andheir presence is a poor prognostic factor in manyancers.20-24 Tregs can also play a beneficial role as they areital in the induction of tolerance to transplant allografts.25

he importance of Treg-mediated immunosuppression isppreciated, as such, their role in other states of immuneegulation is expanding. We hypothesize that exposure toBT results in induction of Tregs, and this induction isttenuated with LR and accentuated with prolongedtorage.

ETHODSreparation of blood componentsfter informed consent according to guidelines set forth by

he Colorado Multiple Institutional Review Board, healthydult volunteers donated 1 U whole blood, which was sep-rated into components and stored according to the Amer-can Association of Blood Banks criteria. Fifty percent, byeight, of the PRBCs were LR on day 0 using Pall BPF4

Pall Corporate) and Fenwall-Sepacell R500-ii (Baxter-

Abbreviations and Acronyms

ABT � allogeneic blood transfusionCFSE � carboxyfluorescein diacetate succinimidyl esterCtl � control mediaIL � interleukinLR � leukoreducedNLR � nonleukoreducedPBMC � peripheral blood mononuclear cellPRBC � packed red blood cellTreg � regulatory T cell

enwall) third-generation filters before routine storage. c

amples from the LR and nonleukoreduced (NLR) PRBCsere drawn from sterile couples on days 1 and 42 routine

torage from the same donor. The plasma fraction (super-atant) was isolated from each of these PRBC samples byentrifugation at 5,000g for 7 minutes followed by an ad-itional spin of 12,500g for 5 minutes to remove acellularebris. Each supernatant was aliquoted and stored at70°C until later use. PRBC units were washed poststor-

ge using a Cobe 2991 Cell Processor (Gambro BCT) perublished methodology, and the plasma fraction (superna-ant) was isolated, aliquoted, and frozen using centrifuga-ion at g-forces recommended for plasma isolation perABB criteria, with a final spin at 12,500g to remove acel-

ular debris.26-29

eripheral blood mononuclear cell isolation andlood component exposureeripheral blood was collected from healthy human volun-eers, and peripheral blood mononuclear cell (PBMC) frac-ion was isolated using a Ficoll-Paque density gradientAmersham Biosciences AB). Cells were washed with Dul-ecco’s PBS three times before their use or stored in 10%imethyl sulfoxide solution in control media (RPMI-1640edia supplemented with 100 U/mL penicillin, 100g/mL streptomycin, 2 mmol/L L-glutamine, and 10%ooled human serum; Gemini Bio-Products) at �70°Cntil use.For PRBC supernatant exposure experiments, 2 � 106

solated normal human PBMCs were cultured in 1.5 mLontrol media or 1.5 mL PRBC supernatant at 20% con-entration by volume in control media per well on a 24-ell plate (Corning) with or without 1 �g/mL solublenti-CD3 antibody (BD Biosciences) at 37°C in 5% CO2

or 5 days. In other experiments, PBMCs were cultured inarious concentrations of PRBC storage solution (AS-5,hich contains dextrose, adenine, mannitol, and sod-

um chloride; and the anticoagulant-preservative solu-ion CPD, which contains citrate, dextrose and monobasicodium phosphate) or washed PRBC supernatant also at0% concentration by volume in control media. PRBCupernatant from a single donor was collected and pro-essed for LR, NLR, storage duration, plasma fraction-tion, and washing, as described for use in each single ex-eriment. To control for variability among PRBC andBMC donors, different PRBC and PBMC donors weresed for every repetition of each experiment up to 10epetitions.

PBMCs were then harvested, stained for 15 minutes atoom temperature with murine antihuman CD4 antibodyTC-conjugated; Invitrogen), murine antihuman CD25ntibody (fluorescein isothiocyanate�conjugated; BD Bioscien-
es), and fixed with commercial fixation/permeabilization

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112 Baumgartner et al Stored RBC Transfusion Induces Regulatory T Cells J Am Coll Surg

olution according to manufacturer’s instructionseBioscience). PBMCs were then incubated for 30 minutes atoom temperature with normal rat serum in permeabiliza-ion buffer (eBioscience) to prevent nonspecific bindingnd with rat antihuman Foxp3 antibody (phycoerythrin-onjugated; eBioscience). PBMCs were then washed inermeabilization buffer and resuspended in 1% parafor-aldehyde in Dulbecco’s PBS and stored at 4°C in the

ark until flow cytometric analysis was performed. Cellsere analyzed by flow cytometry using a FACSCaliber

Becton Dickinson) with gating for lymphocytes, thenD4� T cells, then on Foxp3� T cells as Tregs, or alterna-

ively on CD25�Foxp3� T cells as activated (non-Treg) Tells. Flow data were analyzed using the FlowJo softwarerogram (Tree Star). The Foxp3 gate was established usingD4� cells as Foxp3� controls and was identical for each

ample per group. Appropriate isotype control antibodiesere used to control for nonspecific staining (Invitrogennd eBioscience).

reg suppression assayormal human PBMCs were isolated and cultured in con-

rol media or 20% PRBC supernatant with our without 1g/mL soluble anti-CD3 antibody (BD Biosciences) for 5ays as described. The PBMCs were then harvested,ashed with Dulbecco’s PBS, and sorted by the magneticead immunosorting technique (MACS cell separation;iltenyi Biotec) into CD4�CD25� Tregs and CD4�

D25� T-responder cells. Cells were first sorted intoD4� isolates by negative selection, then into CD25� orD25� isolates by positive selection according to the man-facturer’s instructions.Sorted Tregs and T-responder cells were stained with�M carboxyfluorescein diacetate, succinimidyl ester

CFSE) for 15 minutes at 37°C (Invitrogen). The 5 � 104

orted CD4� T-responder cells were incubated with either� 104 additional CD4� T responder cells with or with-

ut 3 �g/mL soluble anti-CD3 and 3 �g/mL soluble anti-D28 activating antibodies (BD Biosciences), or with 5 �04 sorted Tregs from control media, PRBC supernatantxposure, control media with anti-CD3 stimulation orRBC supernatant with anti-CD3 stimulation, with 3g/mL soluble anti-CD3 and 3 �g/mL soluble anti-CD28

ntibodies in 300 �L control media in a 96-well roundottom plate (Corning) at 37°C in 5% CO2.All cells were harvested after 5 days, washed in Dulbec-

o’s PBS, and stained with murine antihuman CD4 an-ibody (allophycocyanin-conjugated; Invitrogen) asescribed. Cells were then washed and fixed using com-ercial fixation/permeabilization solution as described

eBioscience). Cells were then washed and resuspended in
% paraformaldehyde in Dulbecco’s PBS and stored at a
°C in the dark until flow cytometric analysis was per-ormed. Cells were acquired on a FACSCaliber flow cy-ometer (Becton Dickinson). Flow data were analyzed us-ng the FlowJo software program (Tree Star) with gatingased on forward versus side scatter for live lymphocytes,hen on CD4� T cells, then on CFSE with the proportionf divided cells estimated as the population of cells withiluted CFSE signal compared with the undivided CFSEhigh

opulation. Negatively or singly stained cells were used asompensation controls and the appropriate isotype con-rols for nonspecific staining (Invitrogen).

ytokinesupernatants from the PRBC supernatants and fromRBC supernatant exposure experiments were collected,entrifuged at 1,000g to remove acellular debris, andnalyzed for interleukin (IL)-1�, IL-2, IL-4, IL-10,nterferon-�, and tumor necrosis factor�� by multiplexead array according to manufacturer’s instructions (Bio-ad). Total transforming growth factor��1 levels wereetected by standard ELISA (ELISATech).

tatisticstatistical analyses were performed using JMP softwareackage version 5.0.1 for Windows (SAS Institute). Resultsre expressed as mean values � SEM unless otherwise in-icated. For comparisons of three or more groups, ANOVAas used and a p value � 0.05 was considered statistically

ignificant.

ESULTSRBC supernatant induces Tregs

o determine if PRBC supernatant induces Tregs, andf storage or LR might alter this effect, normal humanBMCs from healthy volunteers were exposed to control me-ia alone (Ctl) or the acellular plasma fraction from PRBCPRBC supernatant) units from a single donor stored for 1-ay or 42-day LR or NLR at 20% concentration by volume

n Ctl for 5 days. One-day and 42-day supernatants and LRnd NLR supernatants were collected from the same PRBConor per group. Twenty-percent concentration was se-

ected as a clinically relevant amount to approximate a pa-ient transfused approximately 10 U PRBCs. PBMCs werenalyzed by flow cytometry after exposure for the percent-ge of Foxp3� Tregs in all CD4� T cells. Without anytimulus, the PRBC supernatant did not alter Treg induc-ion (1.7% � 0.2%, 1.2% � 0.3%, 1.4% � 0.2%, and.9% � 0.3% Foxp3� Tregs per CD4� T cells for 1-dayLR, 42-day NLR, 1-day LR, and 42-day LR, respec-

ively) compared with Ctl (1.3% � 0.3%; Fig. 1). With
nti-CD3 stimulation, exposure to PRBC supernatant

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ore than doubled the percentage of Tregs compared withtl plus anti-CD3, and prolonged storage or LR did not

lter this induction (6.2% � 0.6%, 6.0% � 0.6%, 5.6% �.5%, and 5.9% � 0.4% for 1-day NLR, 42-day NLR,-day LR, and 42-day LR, respectively, versus 2.7% �.6% for Ctl; p � 0.05 for all groups versus Ctl, n � 10ndependent experiments with 10 different PRBC donors;ig. 1). In sum, PRBC supernatant, regardless of durationf storage or LR, induces Foxp3� Tregs from PBMCs stim-lated with anti-CD3.

RBC supernatant does not alter T-cell activationecause anti-CD3 stimulation itself has been reported toause nonspecific Foxp3 expression,30 the ability of PRBCupernatant to nonspecifically activate T cells was deter-ined. PBMCs were exposed to Ctl or one of the fourRBC supernatants used previously (1-day NLR, 42-dayLR, 1-day LR, and 42-day LR) with or without anti-D3 stimulation. PBMCs were then analyzed by flow cy-

ometry for the percentage of activated CD25� T cellsmong all CD4� T cells. Because most Tregs also expressD25, an attempt was made to exclude this population byating on the population of CD25�Foxp3�CD4� T cellss activated, non-Treg, T cells. Without stimulation, theercentage of activated T cells was low in all groups (4.1% �.7%, 5.8% � 1.7%, 2.6% � 0.5%, 6.2% � 1.1%, and.3% � 0.6% CD25�Foxp3� activated T cells per CD4�

cells for Ctl, 1-day NLR, 42-day NLR, 1-day LR, and2-day LR, respectively; Fig. 2). With anti-CD3 stimula-ion, the percentage of activated T cells increased in allroups. PRBC supernatant did not activate more T cellshan Ctl and even nonsubstantially activated slightly lesshan control (52% � 13%, 53% � 13%, 56% � 8%, and9% � 9% for 1-day NLR, 42-day NLR, 1-day LR, and2-day LR, respectively, versus 75% � 10% for Ctl; n � 4;

igure 1. Regulatory T cells (Tregs) are induced from stimulatederipheral blood mononuclear cells by packed red blood cell super-atant. Packed red blood cells were exposed to control media (Ctl)r packed red blood cell supernatant after 1 day or 42 days oftorage (D1 or D42), with leukoreduction (LR) or without leukore-uction (NLR) � anti-CD3 stimulation. Compiled data from 10 inde-endent experiments expressed as mean � SEM. *p � 0.05 versustl � anti-CD3 group.

ig. 2). Taken together with the Treg results, these data c

uggest PRBC supernatant specifically induces Tregs, butoes not result in global T-cell activation, and this induc-ion is not altered by storage or LR.

RBC-induced Tregs are immunosuppressiveo characterize the suppressive capacity of PRBC-inducedregs, the ability of these Tregs to suppress the proliferationf CD4�CD25� T-responder cells was assessed. After ex-osure to Ctl or one of the PRBC supernatants (42-dayR) with or without anti-CD3 stimulation for 5 days,D4�CD25� Tregs and CD4�CD25� T-responder cellsere sorted by magnetic bead immunosorting. Tregs were

orted based on their CD25 expression rather than byoxp3 as only surface markers can be used to retain viableells. Figure 3A confirms what others have previouslyhown, that sorting CD4�CD25� T cells enriches foroxp3� cells.31 Tregs induced by PRBC or PRBC � anti-D3 stimulation (PRBC Tregs or PRBC � Stim. Tregs)

uppressed proliferation of isolated CD4�CD25�

-responder cells to a similar degree as control or control �nti-CD3 stimulation Tregs (Ctl Tregs or Ctl � Stim. Tregs)arvested from Ctl (resulting in 15% � 7%, 11% � 5%,0% � 7%, and 8% � 2% divided CD4� T cells for Ctl,RBC, Ctl � Stim. and PRBC � Stim. Tregs, respectively,ersus 34% � 9% in wells without Tregs, p � 0.05, n � 4;ig. 3C). PRBC � Stim. Tregs maintained their suppressiveapacity when diluted to a 3:1 T-responder cell to Treg

atio, resulting in only 8% � 6% of divided CD4� T cellsFig. 3C). Tregs induced by PRBC suppressed proliferationf T-responder cells. PRBC � Stim. Tregs also were anergichen cultured alone with stimulation (Fig. 3C) and 74% �% express CD25, consistent with the reported in vitroharacterization of Tregs.

RBC supernatant does not substantially alterytokine responseo identify possible cytokines in PRBC supernatant that

igure 2. T-cell activation is not altered by packed red blood cellupernatant. Peripheral blood mononuclear cells were exposed toontrol media (Ctl) or packed red blood cell supernatant after 1 dayr 42 days of storage, with leukoreduction (LR) or without leukore-uction (NLR) � anti-CD3 stimulation. Compiled data from four

ndependent experiments expressed as mean � SEM.

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ytokine profile including IL-1�, IL-2, IL-4, IL-10,nterferon-�, tumor necrosis factor��, and transformingrowth factor��1 levels were evaluated in the PRBC su-ernatant by cytokine bead array or standard ELISA. Be-ause other investigators have reported that LR diminisheshe cytokine content of PRBCs,7,11 and because both LRnd NLR supernatants induced Tregs to a similar degree,nly LR PRBC supernatants from 1-day and 42-day werevaluated for cytokines still present after prestorage leuko-yte filtration. Levels of all seven cytokines were detectablen the 1-day LR PRBC supernatant, but were reduced afterrolonged storage of 42 days, reaching statistical signifi-ance for all cytokines except transforming growth fac-or�� for the small number of supernatants assayed (n �; p � 0.05; Fig. 4A).To characterize additionally the immune response of

BMCs to PRBC exposure, the same cytokines were exam-ned in the supernatants of the PRBC supernatant exposurexperiments. Namely, we sought to determine if PRBCupernatant resulted in an immunosuppressive cytokineesponse to correlate with Treg induction. Supernatantsrom the previous PRBC supernatant exposure experi-ents were collected and analyzed for IL-1�, IL-2, IL-4,

L-10, interferon-�, tumor necrosis factor��, and trans-orming growth factor��1 levels. Anti-CD3 stimulationncreased the levels of most cytokines in the supernatantFig. 4). IL-1�, IL-4, interferon-�, tumor necrosisactor��, and transforming growth factor�� levels wereo different in supernatants from PRBC-exposed PBMCsompared with control PBMCs (Fig. 4). IL-2 levels werelightly higher from anti�CD3-stimulated PBMCs ex-osed to PRBC supernatants versus Ctl (13 � 2 and 12 �for 1-day LR and 42-day LR, respectively, versus 7 � 1

g/mL for Ctl; p � 0.05, n � 3; Fig. 4), but had slightlyower IL-10 levels (186 � 24 and 137 � 23 for 1-day LRnd 42-day LR, respectively, versus 322 � 53 pg/mL fortl; p � 0.05, n � 3; Fig. 4). Taken together, PRBC

upernatant did not substantially alter levels of IL-1�, IL-4,nterferon-�, tumor necrosis factor��, and transformingrowth factor�� secreted from PBMCs and only slightlyncreased IL-2 levels and decreased IL-10 levels.

RBC storage solution does not induce Tregs butashed PRBC supernatant abrogates Treg inductionecause all PRBC supernatants induced Tregs regardless of

torage time or LR, a common mediator of Treg inductionn all supernatants was sought. One component present inll supernatants is the storage solution of the PRBCs. Thisontains the additive solution AS-5, which contains dex-rose, adenine, mannitol, and sodium chloride; and thenticoagulant-preservative solution CPD, which contains
itrate, dextrose and monobasic sodium phosphate.26 p
BMCs were exposed to various concentrations of storageolution or Ctl, with or without anti-CD3 stimulation for

days. PBMCs were analyzed for Foxp3� Tregs andD25�Foxp3� activated T cells as in previous experi-ents. Concentrations of storage solution � 0.5% ap-

eared to globally reduce cell numbers after 5 days, andoncentrations � 5% did not result in sufficient numbersf cells to analyze by flow cytometry. At 0.5% concentra-ion, the storage solution did not induce Tregs with (4.8% �.9% Foxp3� Tregs among all CD4� T cells) or without1.3% � 0.1%) anti-CD3 stimulation compared with Ctl3.4% � 2.0% and 1.2% � 0.2%, for Tregs with or with-ut anti-CD3, respectively). The storage solution also didot alter the percentage of activated T cells with and with-ut stimulation versus control. Storage solution alone doesot reproduce the effects of the whole PRBC supernatant

n terms of Treg induction.To evaluate the effects of the plasma fraction of PRBC

upernatant, units were washed poststorage with saline be-ore supernatant collection to reduce plasma content andxposed to PBMCs as in previous experiments. Usingashed PRBC supernatant from fresh units (day 7) or oldnits (day 42), induction of Tregs in PBMCs stimulatedith anti-CD3 was abrogated and was no different than intl (2.1% � 1.2% and 1.7% � 0.8% versus 2.4% � 1.4%

or 7 days and 42 days washed PRBC supernatant versustl; Fig. 5A). Washed PRBC supernatants also did not alter-cell activation versus Ctl (Fig. 5B). Removal of thelasma fraction of PRBCs abrogates the inductive effect onregs. In addition, levels of transforming growth factor��1ere undetectable in fresh and stored washed PRBC

upernatant.

ISCUSSIONransfusion-related immunosuppression is potentially me-iated by any component of a PRBC transfusion, includ-

ng erythrocytes and transfused leukocytes, platelets, andoluble factors derived from these cells or originally presentn the donor plasma. The current study was undertaken tonvestigate the role of soluble factors in transfusion-relatedmmunosuppression. Namely, induction of Tregs was ex-mined as a component of immunosuppression from anBT. We found that PRBC supernatant was able to induceregs from stimulated PBMCs. This induction occurred,

omewhat surprisingly, to a similar degree regardless of LRr duration of storage. The PRBC supernatant did notlobally activate T cells, and the supernatant specificallypregulates Tregs. Tregs induced by the PRBC supernatantre immunosuppressive, as they suppress proliferation ofesponder T cells. PRBC-induced Tregs are at least as sup-
ressive as control Tregs and even showed a trend of greater


Figure 3. Regulatory T cells (Tregs) induced by packed redblood cell (PRBC) supernatant are immunosuppressive. Pe-ripheral blood mononuclear cells were exposed to controlmedia (Ctl) or PRBC supernatant � anti-CD3 stimulation. (A)Peripheral blood mononuclear cells were then sorted by mag-netic beads into Tregs (CD4�CD25�) or T-responder cells(CD4�CD25�). CD4�CD25� sorted T cells are enriched forFoxp3� cells as depicted in the flow plot. Sorted cells werestained with carboxyfluorescein diacetate succinimidyl ester(CFSE) and T-responder cells from Ctl were then coculturedwith Tregs from control media (Ctl Tr), PRBC supernatant(PRBC Tr), control media with anti-CD3 (Ctl Stim. Tr) or PRBCsupernatant with anti-CD3 (PRBC Stim Tr) in various ratios �anti-CD3/anti-CD28 stimulation for 5 days. (B) Representa-tive flow plots depict the percentage of divided CD4� T cells asthe CFSE diluted population. (C) Compiled data from four inde-pendent experiments expressed as mean � SEM. *p � 0.05
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uppression. All cytokines evaluated decreased with storagen LR PRBC supernatants, and none were persistently ele-ated after 42 days of storage. In addition, cytokine levelsrom PBMCs exposed to the supernatant were not substan-ially different than from control, eliminating the possibil-ty that a proinflammatory cytokine response can counter-alance Treg induction, and also reducing the possibilityhat the activity of the induced Treg is IL-10� or trans-orming growth factor-��dependent, as neither of thesemmunosuppressive cytokines were elevated in the cellularupernatants. The PRBC storage solution itself did notnduce Tregs and washed PRBC supernatant abrogated thenductive capacity of PRBC supernatant on Tregs, implicat-ng the plasma fraction.

The mechanism of immunosuppression after ABT re-ains uncertain. Ghio and colleagues12 have described

unctional immunologic defects, including attenuation ofytotoxicity and decreased mixed lymphocyte reaction af-er PRBC supernatant exposure. These investigators foundhe immunosuppression of PRBC supernatant was amelio-

Figure 4. Cytokine levels in leukoreduction (LR) packed red blood ceblood mononuclear cells to PRBC supernatant does not result in sanalyzed by bead array or standard ELISA (for transforming growth fahad statistically significant decreases in all cytokines except for TGF-(Ctl) or PRBC supernatant from leukoreduced units after 1 day osupernatant was collected and cytokines were measured. Compiled*p � 0.05 versus Ctl � anti-CD3 group.

ated with LR and that the degree of immunosuppression f

orrelated with the concentration of soluble HLA class Iolecules and Fas ligand, which both increased with stor-

ge duration and were reduced with LR. In the presenttudy, storage duration and LR did not alter induction ofregs, so Fas ligand or HLA class I molecules are likely not

nvolved in PRBC-mediated Treg induction. In addition,regs are known to be particularly susceptible to Fas

igand�mediated apoptosis32 and yet, interestingly, theyere upregulated in response to PRBC exposure. PRBC-ediated Treg induction occurred only in the presence of

nti-CD3 stimulation, suggesting the “two-hit” phenome-on described in other aspects of transfusion-related im-unomodulation,2 with T-cell receptor activation andRBC exposure required for Treg induction.Other investigators have suggested a role for Tregs in

ransfusion-mediated immunosuppression, but this hasot been studied directly.33,34 Lapierre and colleagues35 ex-mined aspects of immunosuppression and tolerance afterLR versus LR PRBC transfusion in cancer patients. In

his small, randomized, controlled trial of 35 patients, they

BC) supernatant decreases with storage and exposure of peripheralantial cytokine alterations. (A) LR PRBC supernatant (n � 3) was[TGF]��1) for cytokine concentrations. D42 LR PRBC supernatants) Peripheral blood mononuclear cells were exposed to control mediadays of storage (D1 LR or D42 LR) � anti-CD3 stimulation. Thefrom three independent experiments expressed as mean � SEM.

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ound that the median gene expression of Foxp3 did not

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ncrease 3 days after blood transfusion and the relativehanges were no different from NLR versus LR transfu-ions. In the present study, Treg induction was examinedn vitro using Foxp3 protein expression and suppressiveunction with 5 days of ABT exposure, as Treg inductions time-dependent and can require 4 to 5 days forpregulation.36

The immunosuppressive capacity of ABT to induceregs, regardless of LR or storage duration, was unexpected.rolonged storage has been shown to increase mortality inransfused recipients, most recently reported in a large ob-ervational study of nearly 20,000 cardiac surgery patientsith higher mortality found in individuals who receivedlood older than 14 days.37 The mechanism of this worserognosis remains unclear and it might be the result ofroinflammatory or immunosuppressive effects of theBT. Most investigations of the “storage lesion” of PRBCsave identified increased proinflammatory mediators withrolonged storage.38,39 Prestorage LR has been imple-ented universally in Canada and Western Europe and, to

ome degree, in the US, partly in an attempt to amelioratehe immunomodulation of transfused leukocytes oreukocyte-derived products. The only proved benefits of

igure 5. Washed PRBC supernatant does not induce regulatory Tells (Tregs) or activate T cells. Peripheral blood mononuclear cellsPRBC) were exposed to control media (Ctl) or washed packed redlood cell supernatant after 7 days or 42 days of storage � anti-CD3timulation. Compiled data from three independent experimentsxpressed as mean % (A) Foxp3� Tregs or (B) CD25�Foxp3� acti-ated T cells of all CD4� T cells � SEM.

restorage leukocyte filtration are the reduction of nonhe- d

olytic febrile transfusion reactions, reduction in cyto-egalovirus transmission, decreased HLA alloimmuniza-

ion with platelet refractoriness, and improved short-termurvival in cardiac surgery patients.40,41 Similar to the ef-ects of stored blood transfusions on cardiac patients, it isnclear if this improved survival with LR transfusion is aesult of alterations of immune upregulation or suppres-ion. Although pretransplantation ABT improves allografturvival,42 no human studies have proved prestorage leu-oreduction ameliorates this effect. Similarly, the effects ofeukocyte-reduced ABT on infection risk are inconclu-ive.43 Although animal models have suggested thatrestorage leukoreduction attenuates the protumor effectf ABT,44 no human, randomized, controlled, clinical trialsave been completed to compare LR versus NLR ABT. Theinding that LR does not alter PRBC induction of Tregs

ight be consistent with the lack of clinical evidence of aenefit of LR, and suggests that LR might not attenuateransfusion-related immunosuppression. In addition, theurrent study found that washing the plasma out of theRBC supernatant abrogated its ability to induce Tregs.he plasma fraction contains one or several potential fac-

ors that can induce Tregs in the recipient. Indeed, transfu-ion with plasma alone in the form of fresh-frozen plasmaas been shown to induce immunologic responses in theecipient, although the role of Treg induction in these re-ponses remain unclear.45

There are some limitations of this study. Although thelasma fraction of the PRBC supernatant is required forreg induction, the precise mediator remains unclear.ransforming growth factor��, in conjunction with IL-2xposure and anti-CD3 stimulation, has been shown toenerate Tregs from naïve CD4�CD25� precursors.36

ransforming growth factor�� is present in the PRBCupernatants tested (in addition to IL-2), and presents aossible mechanism of transfusion-mediated inductionf Tregs. Other potential inducers of Tregs that mighte present in the transfused plasma fraction include

hrombospondin-1, vitamin A metabolites, heme oxygenase-1,nd prostaglandin E2. The degree of PRBC-mediated Treg

nduction appears relatively modest, with an increase frompproximately 3% of all CD4� T cells in Ctl to 6% inRBC-exposed cells. Despite the relative rarity of Tregs,hey are quite potent. Treg dysfunction or absence is asso-iated with severe autoimmune disorders, such as multipleclerosis, rheumatoid arthritis, and immune dysfunction,uch as polyendocrinopathy, enteropathy, and X-linkedIPEX).46-48 Absolute increases of only 2% circulating Tregs

er all CD4� T cells or less is strongly correlated withncreased mortality in patients with cancer and an absolute
ecrease of 2% circulating Tregs is correlated with the like-

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ihood of organ rejection 1 year after transplantation.22,49,50

lthough Foxp3� Tregs were induced, CD25� Tregs weresed in the functional assay and this population might

nclude some activated effector T cells. Unfortunately,here is no method to sort viable humanTregs based on theiroxp3 status. The CD25� sort did enrich for Foxp3� cellsnd these cells were suppressive in the present study. Therecise roles of PRBC-induced Tregs in transfusion-ediated immunosuppression are also unclear. Tregs are

enerally activated or induced by a wide variety of antigens,ncluding self-antigens, and by antigen-independent

echanisms.17 They act through cell-contact�dependentnd cell-contact�independent methods, in part by secre-ion of nonspecific immunosuppressive molecules, such asL-10 and transforming growth factor��,17 although theseytokines were not elevated in PBMCs exposed to PRBCupernatant in the present investigation.

In summary, allogeneic blood transfusion induces Tregs

nd this induction does not appear to be attenuated byeukoreduction or altered by duration of storage. This in-uction is independent of exposure to transfused cells, butependent on exposure to allogeneic plasma. These find-

ngs can provide one mechanism of transfusion-related im-unosuppression seen clinically after ABT, and additional

nvestigation into PRBC-mediated Treg induction can pro-ide insights into attenuating this phenomenon.

uthor Contributions

tudy conception and design: Baumgartner, Banerjee,McCarter

cquisition of data: Baumgartnernalysis and interpretation of data: Baumgartner, Silliman,

Moore, Banerjee, McCarterrafting of manuscript: Baumgartnerritical revision: Baumgartner, Silliman, Moore, McCarter

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Stored Red Blood Cell Transfusion Induces Regulatory T Cells