Kawaguchi 2012 Effect of Liposome e

aor_1269 194..201

Effect of Liposome-Encapsulated Hemoglobin onAntigen-Presenting Cells in Mice

*Akira T. Kawaguchi, *Junko Aokawa, †Yuko Yamada, *Fumiaki Yoshiba, *Shunichi Kato,and †Yoshie Kametani

Departments of *Cell Transplantation and Regenerative Medicine and †Immunology, Tokai University School of Medicine,Isehara, Kanagawa, Japan

Abstract: Liposome-encapsulated hemoglobin (LEH) isremoved from the circulation and degraded in the reticu-loendothelial system, including dendritic cells (DCs) andmacrophages. Therefore, LEH at a large dose may over-load the system, cause a competitive inhibition in antigen-presenting activity, and impair the immune response ofthe host. Changes in cellularity of immunocompetent cellswere monitored serially up to 4 weeks by flow cytometryin wild-type mice receiving 20 mL/kg of LEH, syngeneicred blood cells (RBCs), or saline. DCs were collectedfrom the host spleen 1, 7, and 28 days after receiving thesolution and were cocultured with naïve cluster of differ-entiation 4 T cells from T-cell receptor transgenic mice inthe absence or presence of third-party antigens. After

LEH administration, the cellularity of DCs and macroph-ages in the recipient spleen remained unchanged fromcontrol mice receiving RBCs or saline. While subset popu-lations and costimulatory molecule expressions were dif-ferent, DCs from LEH-administered mice expressed highlevels of interleukin-2 production and helper T-cell acti-vation in response to a third-party antigen and superan-tigens, as did the DCs from control mice receiving RBCsor saline. The results suggest that 20 mL/kg of LEH doesnot greatly alter antigen-presenting activity to third-partyantigens. Key Words: Artificial oxygen carriers—Antigen-presenting cells—Dendritic cells—Macrophages—Phagocytosis—Emergency transfusion—Hemorrhagicshock—Contamination—Immune response.

Artificial oxygen (O2) carriers have been devel-oped as substitutes of red blood cells (RBCs) fortransfusion (1,2). Although vascular adverse eventsobserved in cell-free hemoglobin in clinical trials (3)were reportedly avoided in experimental studiesusing liposome-encapsulated hemoglobin (LEH;TRM-645, Terumo, Tokyo, Japan) (4–7) or hemoglo-bin vesicle (HbV) (8,9), there remain concernsregarding its use as a transfusion substitute becauseof a much shorter retention time than that of RBCs.Circulation half-life (T1/2) of LEH and HbV dependson species as well as doses; it is approximately 1 dayin rodents (4–9) and 2–3 days in primates when

10–20 mL/kg is used (10,11). While LEH is removedfrom the circulation and disposed of by the reticu-loendothelial system (RES) similarly to RBCs, thepossible use of LEH for emergency transfusionbefore identical donor blood becomes available (12)may impose an acute metabolic load of hemoglobinand lipids on RES or antigen-presenting cells(APCs), such as dendritic cells (DCs) or macroph-ages, which plays a pivotal role in the triggering of thehost’s immune defense against third-party antigens.Thus, this raises a serious concern, as hemorrhagicshock patients are often exposed to pathogens ataccidents or surgery causing acute blood loss neces-sitating emergency transfusion (12). In our previousstudy (4), a single injection of LEH at a moderatedose (10 mg/kg) did not largely affect the murine orhuman immune status nor did it interfere with thehost’s early response to bacterial enterotoxins (toxicshock syndrome toxin-1 [TSST-1]), so far as lympho-cyte cellularity and interleukin (IL)-2 producibilitywere concerned. While Sakai and colleagues (2)reported that administration of 20 mL/kg of HbV,

doi:10.1111/j.1525-1594.2011.01269.x

Received August 2010; revised March 2011.Address correspondence and reprint requests to Dr. Akira T.

Kawaguchi, Department of Cell Transplantation and RegenerativeMedicine, Tokai University School of Medicine, Shimokasuya 143,Isehara, Kanagawa 259-1193, Japan. E-mail: [email protected]

Artificial Organs36(2):194–201, Wiley Periodicals, Inc.© 2011, Copyright the AuthorsArtificial Organs © 2011, International Center for Artificial Organs and Transplantation and Wiley Periodicals, Inc.

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similar to LEH, transiently reduced phagocytic activ-ity by 40%, we directly examined the activity of APCsfrom LEH-administered mice using a system thatallows the mimicking of the initial encounter of thesetwo cell types with LEH in vivo. In this study, first, weexamined in vivo changes in an immunocompetentcell population in LEH-treated mice and then theactivation (Fig. 1A) and subpopulation changes insplenic DCs and macrophages. Finally, the activity ofDCs (Fig. 1B) in the absence or presence of third-party antigens was tested in vitro, using cluster ofdifferentiation (CD)4+ T cells (helper T cells) har-vested from newly developed transgenic mice (Fig.1C) so as to increase the sensitivity and specificity in

detecting the antigen-presenting activity of DCs fromrecipient animals.

MATERIALS AND METHODS

Liposome-encapsulated hemoglobinEncapsulation of hemoglobin has been proven to

be effective in avoiding extravasation and directcontact of hemoglobin with vascular structure inLEH (1,4–7) as well as in HbV (2,8,9). Relevant char-acteristics of LEH have been reported. Briefly, LEHis a liposome capsule, measuring 230 nm in meandiameter and contains human hemoglobin purified

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FIG. 1. Activity of antigen-presenting cells. Antigen-presenting cells (APC, A) recognize foreign material (I), phagocytes (II), and presentas a specific antigen (III) on MHC class II surface markers together with costimulator protein (B7). Helper T cell (B) with a specific receptor(TCR) comes to be activated through TCR (signal 1) and costimulator protein pathway (signal 2) to activate helper T cell, producing IL-2and CD25 as a surface marker or IL-2 receptor. Helper T cell (C) from OVA23-3/IL-2-GFP mice can be activated through TCR specificto OVA and produce GFP in plasma together with IL-2 production. Helper T cells may also be stimulated by TSST-1 without any specificantigen. Activation of helper T cells from transgenic mice may be evaluated sensitively and specifically by FACS, gated for GFP (*) as wellas CD25 (*).

ARTIFICIAL O2 CARRIER AND ANTIGEN-PRESENTING ACTIVITY 195

Artif Organs, Vol. 36, No. 2, 2012

from donated blood that was outdated for transfu-sion (1). The liposome capsule was coated withpolyethylene glycol to reduce aggregation and recog-nition by RES, as well as to prolong the circulatoryhalf-life to less than 1 day in mice (4), approximately1 day in rats (5–7), and 2–3 days in nonhuman pri-mates (10,11). In this preparation, inositol hexaphos-phate was coencapsulated to control O2 affinity toP50O2 = 40–50 mm Hg in LEH, lower than that ofrodent RBCs (P50O2 = 30 mm Hg). LEH was sus-pended in saline at a hemoglobin concentration of6 g/dL or 20% of volume, which has reduced viscosity(2 cp) compared with blood (5 cp) and specificgravity close to that of plasma. Phosphate bufferedsaline (PBS) and murine syngeneic RBCs, washedand prepared at a hematocrit of 20%, were used ascontrol solutions.

MiceEight-week-old female wild-type mice (BALB/c)

were purchased from CLEA Japan (Kawasaki, Kana-gawa, Japan). T-cell receptor (TCR) transgenic mice(I-Ad, ovalbumin [OVA]323–339 specific) termedOVA23-3 mice (13), expressing TCR specific for apeptide fragment of OVA (OVA323–339) presented bythe I-Ad class II major histocompatibility complex(MHC) molecule, were maintained on a BALB/cbackground over 16 generations. This OVA23-3mouse was crossed with transgenic mouse strain car-rying 5′ flanking region of IL-2 gene conjugated withenhanced green fluorescent protein (GFP) codingregion (IL-2-GFP mouse, kindly provided by Profes-sor Ellen V. Rothenberg) for at least nine generationsas described previously (14). The offsprings weredetermined as transgene carriers by polymerasechain reaction analysis of genome DNA. TheOVA23-3 transgenic mice, IL-2-GFP mice, and theiroffspring (OVA23-3/IL-2-GFP) were kept under spe-cific pathogen-free conditions in the animal facility ofTokai University School of Medicine (Shimokasuya,Kanagawa, Japan).

Administration of LEH, RBCs, and PBSAll experiments were approved by the institutional

review board of Tokai University School of Medicine.Animals received humane care as required. Femalewild-type BALB/c mice (8~10 weeks old) were anes-thetized with ether and received a single intravenousinjection of LEH (20 mL/kg), washed syngeneicRBCs, or PBS via the tail vein on day 0. SyngeneicRBCs were collected from nontreated naïve BALB/cmice by Ficoll-Conray Lymphosepal (Immuno-Biological Laboratories, Gunma, Japan) and centrifu-

gation (2000 ¥ g, 30 min), washed three times withsaline, and diluted with saline to a final RBC suspen-sion, 20% hematocrit.

Monoclonal antibodies and flow cytometryT cells and B cells were identified by fluorescent-

activated cell sorter (FACS) according to theirexpressions of CD3 (pan T cell), CD4 (helper T cell),CD8 (cytotoxic/suppressor T cell), and CD19 (B cell),respectively. DCs and macrophages were identifiedaccording to their expression of CD11c and CD11b,respectively. T-cell activation was determined byCD25 (T-cell activation antigen). B7-1 (CD80) andB7-2 (CD86) are the most extensively studiedcostimulatory molecules of the B7 family (DC acti-vation antigen, Fig. 1A) (15). Interaction of CD28 onT cells with either CD80 or CD86 has been found toaugment T-cell activation, promote T-cell survival,and enhance IL-2 production (16) (Fig. 1B). Thus,antigen-presenting ability may be evaluated bysimply analyzing the expression of these molecules(B7).

CD4 T cells from OVA23-3/IL-2-GFP miceCD4 T cells (helper T cells) were isolated from a

single-cell suspension of splenocytes from OVA23-3/IL-2-GFP mice by positive selection usinganti-mouse CD4-PerCP.Cy5.5 and goat anti-ratimmunoglobulin G microbeads and magnetic cellsorting system columns (Miltenyi Biotec, Gladbach,Germany). OVA23-3/IL-2-GFP mice have a singleTCR repertoire (13), enabling the activation of,ideally, all T cells by a specific antigen, OVA. More-over, the activation and production of IL-2 of mouseT cells can be detected by identifying GFP by flowcytometry (Fig. 1C) instead of measuring IL-2 in thesupernatant (Fig. 1B). In the current study, therefore,both the IL-2 production and the expression ofCD25, an activation marker, by helper T cells weredetected by flow cytometry without damaging thecells. These characteristics allow amplified, sensitive,and specific evaluation of the helper T-cell activationprocess. The purity of CD4+ cells isolated by thismethod was above 95%, as confirmed by flowcytometry.

DCs from BALB/c mice 1, 7, and 28 daysafter administration

After collecting blood, BALB/c mice were sacri-ficed 1, 7, and 28 days after intravenous administra-tion, and the whole spleen was excised and weighed.Spleen cells were isolated by cutting spleens into

A.T. KAWAGUCHI ET AL.196


pieces and digesting them in 5 mL of Collagenase D(Roche, Basel, Switzerland) at 37°C for 30 min. Thecells were washed, the RBCs lysed by incubation for5 min in ammonium chloride buffer, pH 7.2, washedagain, and resuspended in complete Roswell ParkMemorial Institute medium (RPMI) and 10% fetalcalf serum (FCS). The data were from three experi-ments, each using pooled spleen cells from three miceper group. The total number of spleen cells wascounted. CD11c+ DCs obtained from the spleensof BALB/c mice from the three groups (LEH, RBC,and PBS) were stained with anti-mouse CD11c-fluorescein isothiocyanate (FITC) and anti-FITCmicrobeads and then positively isolated by automaticmagnetic cell sorting system. CD11c+ cells, as well asCD11b+, were gated for subset flow cytometry.

StimulationSplenic DCs (1.2 ¥ 105) purified from BALB/c

mice administered with LEH, RBCs, or PBS wereincubated with CD4 T cells (6 ¥ 105) from OVA23-3/IL-2-GFP mice in 48-well flat-bottom platesin a final volume of 200 mL. These coculturedcells were maintained at a density of 2.5 ¥ 106/mLcells in RPMI, supplemented with 10% heat-inactivated FCS (Invitrogen, Carlsbad, CA, USA),160 mg/mL gentamicin, 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES, Sigma-Aldrich, St. Louis, MO, USA), 2 mM glutamine, and50 mM 2-mercaptoethanol at 37°C in 5% CO2. Eachwell was stimulated with OVA (1 mg/mL, Sigma-Aldrich), TSST-1 (1 mcg/mL, Toxin Technology, Inc.,Sarasota, CA, USA), or PBS at 37°C for 24 or 48 h.After the stimulation, the cells were harvested,stained with anti-CD4-PerCP-Cy5.5, and analyzedby FACSVantage to check their purity. Cells withmore than 90% purity were used for the assay ofantigen-presenting ability of T-cell activation andIL-2 production (Fig. 5A); expressions of CD4, GFP,and CD25 were assessed by flow cytometry in thelymphoid gate. GFP expression was monitored byfluorescence 1 channel (Fig. 5B).

Statistical analysisData are expressed as mean � standard error.

The value obtained from LEH- or RBC-administered mouse spleen cells divided by thecomparable value from PBS-treated mice wasshown as mean index � standard deviation (SD).The statistical significance of differences betweentwo groups was analyzed by Student’s t-test, andmultiple comparisons were performed using analysisof variance. A P value less than 0.05 was consideredstatistically significant.

RESULTS

Cellularity of immunocompetent cells inBALB/c mice

The kinetics of the immunocompetent cell ratioobtained from LEH-treated BALB/c mouse spleenswas assayed by flow cytometry. Raw data weredivided by comparable values from PBS-treated miceand shown as mean index � SD (Fig. 2). The ratiostayed around 1 in any of the categories of pan T cells(CD3) and B cells (CD19), helper T cells (CD4), andcytotoxic T cells (CD8), DCs (CD11c), and macroph-ages (CD11b), showing no consistent or significantchanges in prevalence 1, 7, 14, and 28 days afteradministration of LEH (20 mL/kg), as compared withthe same administered volume of PBS. However, thetotal number of spleen cells of LEH-treated miceincreased to 1.8-fold compared with control micereceiving saline 1 day after LEH administration.

Cellularity of APCs in spleenThe percentage of total CD11c+ DCs from spleen

did not significantly change until 28 days after LEHadministration (Fig. 2). The prevalence of DCs (Fig.3A) and their subsets in spleen was analyzed 24 hafter administration, and was expressed as indices toequivalent changes occurring in PBS-treated mice.While the value of the DC’s index was similarbetween LEH and RBC administered to mice after24 h (Fig. 3A, left), the percentage of CD11c high-cell

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FIG. 2. Cellularity of splenic immunocompetent cells. There wasno significant or consistent change in the percentages of immu-nocompetent cells; between pan T cell (CD3) and B cell (CD19),between T cell subsets (CD4 vs. CD8), and between DCs(CD11c) and macrophages (MOs, CD11b) in spleen analyzedsequentially using flow cytometry.



fraction (CD11chigh, mainly including myeloid DCs orconventional DCs) declined significantly to 60% ofthe total CD11c+ DCs found in spleens of RBC-administered mice (Fig. 3A, middle). Meanwhile, theCD11c intermediate cell fraction (CD11cint, mainlyincluding plasmacytoid DCs) tended to be higher inthe presence of LEH, although the difference did notachieve significance (P = 0.075). The percentage oftotal CD11b+ cells, mostly macrophages, from spleendid not significantly change 1, 7, and 28 days afterLEH administration (data not shown), although ittended to be lower in the presence of LEH. Subsetsof macrophages were shown 1 day after administra-tion (Fig. 3B); the percentage of CD11bhigh cell frac-

tion (Fig. 3B, middle) and CD11b intermediate cellfraction (CD11bint, Fig. 3B, right) tended to be lowerin the presence of LEH, but significance was notachieved (P = 0.13).

Expression of costimulatory molecules onCD11c+ DCs

DCs up-regulated CD86 expression after adminis-tration of LEH compared with RBCs (Fig. 4A). Thedifference in CD86 expression was significant atday 1, became less prominent at day 7, and returnedto equivalence 28 days after administration betweenthe LEH- and RBC-transfused groups. Costimula-tory CD86 expression on the CD11chigh subset was

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FIG. 3. Percentages of cells expressing CD11c+ and CD11b+. Percentages of CD11c+ cells (DCs, A) as well as CD11b+ cells (MOs, B)in spleens analyzed 24 h after 20 mL/kg of intravenous administration of LEH, RBC, or PBS. Data were from three experiments, eachcontaining three mice per group. Asterisk (*) depicts P < 0.05 as compared with RBC-treated group.

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FIG. 4. Expression of costimulatory molecules on CD11c+ DCs. Expression of costimulatory molecules (CD86) on spleen DCs (A) 1, 7,and 28 days after administration of LEH or RBC compared with PBS-treated mice as control. Spleen cells were stained with anti-CD86and anti-CD11c, and frequency and intensity were measured by FACS gating on CD11c+ cells. Those values in LEH- or RBC-administered mice 24 h after administration were obtained for CD11c high cell subset (CD11chigh) or CD11c intermediate cell subset(CD11cint) and presented as percentage of CD86-expressing cells (B) using PBS-treated mouse spleens as standard.



highly enhanced (P < 0.01), but the CD86 expressionon CD11cint was significantly attenuated (P < 0.04)1 day after administration (Fig. 4B). In contrast,costimulatory CD80 expression on the total CD11c+DCs transiently declined to 40% of DCs from RBC-transfused mice, while CD11c + CD80 high cellsbecame predominant in LEH-treated mice comparedwith RBC-transfused mice 7 days later (data notshown).

IL-2 production and CD25 expression asAPC activity

The frequency of GFP+ cells was assessed 24 and48 h after stimulation with OVA peptide or TSST-1(Fig. 5A, center) in the coculture of naïve CD4+ Tcells from OVA23-3/IL-2-GFP transgenic mice (Fig.5A, right) incubated with DCs purified from pooled

LEH-, RBC-, and PBS-administered mouse spleens(Fig. 5A, left). The expressions of CD4, GFP, andCD25 were assessed by flow cytometry in the lym-phoid gate (Fig. 5B).The FACS profiles are represen-tative of coculture from 24 h postantigen stimulation,presenting the proportion of CD4+ cells comprisingIL-2-GFP (indicating IL-2 expression) and CD25expression (indicating T-cell activation). In all cases,10–17% of CD4 T cells up-regulated GFP expressionirrespective of the origin of DCs, and 60–70% of CD4T cells up-regulated CD25 in all culture conditionswith antigen stimulation. There was no differencebetween coculture durations of 24 and 48 h. In threeexperiments, the average of cell population double-positive for GFP as well as CD25 was similarbetween mice infused with RBC or LEH, irrespectiveof the presence of OVA or TSST-1 (Fig. 5C). Therewas no significant deviation from the ratio 1 or

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FIG. 5. DCs from LEH-administered spleen. Three DC populations purified from pooled LEH-, RBC- and PBS-administered mousespleens were cocultured with naïve CD4 T cells harvested from OVA23-3/IL-2-GFP transgenic mice without (-) or with the presence ofovalbumin (OVA) or TSST-1 (TSST-1). The expressions of CD4+, GFP+, and CD25+ cells were assessed by flow cytometry in thelymphoid gate, and the frequency of IL-2/GFP+ cells was assessed 24 h after stimulation (A). Values in the plots represent the proportion(%) of the CD4+ gate that IL-2-GFP and CD25+ comprise (B). The percentage of dual positive cells (C), average of three experiments,revealed that DCs from spleens 24 h following LEH administration showed no impairment in their ability to activate helper T cells ascompared with the mice administered with RBC.



difference between the groups either 7 or 28 daysafter infusion (data not shown).

DISCUSSION

Clinical needs for transfusion often derive fromtrauma and/or surgery, involving concurrent expo-sure to pathogens and/or tumor antigens. We havebeen using type O Rh (+)-washed and -packed RBCsfor emergency transfusion for patients with hemor-rhagic shock before identical RBCs become available(12). Although a shortened ischemic period has beendemonstrated to be advantageous in terms of survivalover the risk of minor mismatch transfusion (12),the volume of transfusion derived from a 400-mLpackage amounted to 3.05 � 1.20 units of washedand packed RBCs. While this amount does not poseany concern in the case of RBCs, the shorter vascularretention time of LEH may raise the potential riskthat LEH would overload the RES and attenuate theantigen-presenting activity of the host. APCs take upand process antigens and present them to T lympho-cytes with the help of MHC molecules as the initialtrigger of adaptive immune response (Fig. 1A),inducing either tolerance to self-specific peptides orimmunity against foreign antigens (Fig. 1B). In thecurrent study, the antigen-presenting ability to third-party antigens was evaluated in the presence orabsence of LEH 20 mL/kg administration, a doseequivalent to that of emergency transfusion (12).

We previously reported that 10 mL/kg of LEHadministration induced no obvious change of cellu-larity in T cell subsets and B cells in murine, as well asin the reconstituted human immune system in thefirst 4 weeks postadministration (4). Also in thecurrent study after the administration of 20 mL/kg ofLEH (Fig. 2), the population of immunocompetentcells, including DCs and macrophages, in the periph-eral blood or spleen did not differ significantly fromthe changes occurring in mice treated with PBS. Sple-nomegaly was observed in recipient mice transfusedwith LEH; the 1.8-fold increase in the total numberof cells in the spleen 1 day after LEH infusion wascomparable with the reported degree of splenom-egaly after HbV infusion (2). As the spleen is themain organ to dispose of LEH or HbV, accumulationof such foreign materials may induce the migrationof other immunocompetent cells or scavengers,although a bias of cellularity was not detected(Fig. 2).

The CD11c expression level is reported to be dif-ferent between myeloid DCs and plasmacytoid DCs(17,18). Therefore, it is impossible to clearly identifywhich subset of DCs or macrophages was altered

between the LEH-infused and the RBC-transfusedmice. While the total cellularity of CD11c+ DCsremained unchanged, the fraction of CD11chigh DCs(myeloid DCs) was significantly reduced and thefraction of CD11cint DCs (plasmacytoid DCs) tendedto be increased (Fig. 3A), indicating a decrease ofconventional DCs or an increase of plasmacytoidDCs. Similarly, CD11b is expressed not only on mac-rophages but also on granulocytes (19) and naturalkiller cells (20). Macrophages were not differentbetween the LEH- and RBC-transfused mousespleens (Fig. 3B).

A part of the antigen-presenting process involvesthe induction of B7 molecules (Fig. 1A), whichincludes CD80 and CD86 expression and a proin-flammatory cytokine response that helps activateT cells to counteract against the specific antigen(15,16). The expression of B7 family proteins wasup-regulated by the administration of LEH (Fig. 4).Although the human hemoglobin contained in LEHmight have induced xenogeneic immune response inthe recipient mice, it may have been too quick, as theactivation was most prominent 1 day after adminis-tration, decreased in 7 days, and became equivalentto RBC transfusion 4 weeks later (Fig. 4A). There-fore, the early increase in B7 family expression in theLEH-treated mice may indicate, at least, that LEHadministration was not immunosuppressive as inRBC transfusion. Subsets of DCs examined 1 dayafter administration showed significantly differentpatterns of activated antigen (B7) expressionbetween LEH- and RBC-treated mice (Fig. 4B), sug-gesting that conventional myeloid DCs might haverecognized LEH as a foreign material.

IL-2 productivity of helper T cells interacting withDCs from mice infused with LEH was examined toevaluate their antigen-presenting ability. As conven-tional enzyme-linked immunosorbent assay for IL-2determination in culture supernatant may not distin-guish IL-2-producing cells from nonproducing cells,we developed double-transgenic OVA23-3/IL-2-GFPmice in the current study. This transgenic mousestrain makes it possible to analyze living cells, whichexpress GFP under the control of IL-2 promoter andenhancer after OVA or TSST-1 stimulation, provid-ing a new approach for comparing the degree of earlyT-cell activation induced by different APCs. MostGFP + CD4+ T cells express CD25 simultaneouslyafter stimulation, suggesting that IL-2 secretion iscorrelated with the expression of the activationmarker. The CD25+, GFP- cells may include regula-tory T cells, although they might occupy only a part ofthe CD25+ cell population. In the current study, DCsfrom LEH-treated or RBC-transfused mice did not



differ in their ability to support IL-2 production andCD25 expression levels when measured at any of thetime points, 1 day (Fig. 5), 7 days, and 4 weeks follow-ing administration.

Although only a specific antigen (OVA) was testedin the current study, other antigens may induce asimilar response in the host regardless of the pres-ence or absence of LEH, as the primary antigenrecognition process is the same. Similarly, the immu-nologic cascade following TSST-1 stimulation is alsocommon for several pathogens.While the presence ofLEH does not seem to alter the primary immuno-logic response to third-party antigens, the effect ofrepeated administrations of LEH on the secondaryimmune response may need to be tested next forfuture clinical use.

In conclusion, the administration of 20 mL/kg ofLEH did not interfere with antigen presentation byDCs that express high levels of costimulatory pro-teins and are able to activate naïve T cells to produceIL-2 in response to both conventional antigen andbacterial superantigens as efficiently as DCs fromRBC-transfused mice in vitro.Although IL-2 produc-tion and/or T cell activation is one of the earliestevents in the serial immune response, such timing isrelevant when the RES is loaded to the heaviestmetabolic burden and concomitantly exposed tothird-party antigens.Afterwards, a large dose of LEHadministration may possibly induce adverse effectson the later events, including proliferation and/orproduction of other cytokines such as IL-4,interferon-gamma, and IL-17. Therefore, furtheranalyses may become necessary to clearly identifythe effect of large-dose LEH administration on eachevent of the immune response.

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