antigen modulation confers protection to red blood cells...

The Journal of Immunology

Antigen Modulation Confers Protection to Red Blood Cellsfrom Antibody through Fcg Receptor Ligation

Sean R. Stowell,*,1 Justine S. Liepkalns,*,1 Jeanne E. Hendrickson,*,†

Kathryn R. Girard-Pierce,* Nicole H. Smith,* C. Maridith Arthur,* and

James C. Zimring*,‡

Autoantibodies and alloantibodies can damage self-tissue or transplanted tissues through either fixation of complement or ligation

of FcgRs. Several pathways have been described that imbue self-tissues with resistance to damage from complement fixation, as

a protective measure against damage from these Abs. However, it has been unclear whether parallel pathways exist to provide

protection from FcgR ligation by bound Abs. In this article, we describe a novel pathway by which cell surface Ag is specifically

decreased as a result of Ab binding (Ag modulation) to the extent of conferring protection to recognized cells from Fcg-dependent

clearance. Moreover, the Ag modulation in this system requires FcgR ligation. Together, these findings provide unique evidence of

self-protective pathways for FcgR-mediated Ab damage. The Journal of Immunology, 2013, 191: 5013–5025.

Although adaptive immunity has the capacity to recognizea vast range of antigenic determinants, this inherent plas-ticity also increases the risk of autoimmunity (1). As a

result, a distinct set of regulatory mechanisms evolved to aidadaptive immunity in discriminating self from non-self (2, 3).Although central and peripheral tolerance mechanisms reduce thelikelihood of self-reactivity, these regulatory programs occasion-ally fail to fully eliminate or inactivate self-reactive cells (4).When tolerance mechanisms fail, the cellular targets of immunitythemselves can provide an additional layer of protection throughresistance to effector mechanisms. Such cellular protection oftenreflects cell surface expression or release of soluble target cell–derived factors that specifically modulate autoreactive cells (5–7),reducing the level of direct cell-mediated injury. Alternatively,some cell types have intrinsic resistance to the toxic effects of self-effector molecules that otherwise potently destroy many patho-gens (8). Overall, these pathways are best understood in thecontext of cell-mediated immunity [e.g., induction of anergy ordeletion in responding T cells and/or resistance to effector path-ways such as perforin, granzyme, and cytokine-mediated killing(9–12)].In contrast to intrinsic resistance to cell-mediated destruction,

intrinsic resistance to humoral immunity poses a distinct challenge.

Although Abs in this case mediate the tissue damage, the Ab-secreting cells themselves often reside at distant sites from thetarget organ (13). As a result, target tissues possess little, if any,ability to directly modulate the Ab-secreting cells. In such a case,an alternative strategy must be used in the form of resisting theeffects of Abs themselves. This strategy is not thematically dif-ferent from CD8+ T cells resisting perforin- or granzyme-mediatedtoxicity to escape issues of fratricide during cellular immune re-sponses (11). However, as effector pathways for Abs are quitedistinct from those used by T cells, the particular mechanisms mustbe suited to Ab-mediated damage.Abs typically activate one or both of two primary effector mech-

anisms following engagement of Ag (14). Ab-induced comple-ment activation results both in target opsonization (through C3deposition) and in cellular lysis (through membrane attack complexformation) (15). In addition, binding of Ab deposits Fcg domains,which directly opsonize cellular targets through FcgRs (16). In thecase of complement, multiple resistance pathways have been de-scribed by which self-tissues prevent complement-mediated de-struction, including both inhibition of complement activation andfacilitation of degradation of whatever complement may becomeactivated (8, 17, 18).In contrast to the multiple and well-described mechanisms by

which self-tissues avoid damage from complement, it is less clearif there are corresponding pathways to resist the effects of Fcgdomains ligating FcgRs. However, data in multiple systems sug-gest one pathway may exist. In particular, target tissues appear topossess the ability to alter the autoantigen(s) or alloantigen(s)being recognized such that Ab binding is substantially decreasedor eliminated. Such pathways have been observed for nicotiniccholinergic receptors in myasthenia gravis (19, 20), Ags on plate-lets during idiopathic thrombocytopenic purpura (21), and Agson RBCs during autoimmune hemolytic anemia (22). Similarpathways have been observed in response to alloantibodies aswell, which may play a role in transplanted organs “accommo-dating” over time to alloantibodies (23, 24). Finally, certain typesof neoplasia can co-opt the pathways to escape mAb therapy, suchas the selective shedding/shaving of CD20 by chronic lymphocyticleukemia (25). For the purposes of the current report, this generalphenomenon will be termed “Ag modulation” and refers to an Ab-

*Department of Pathology and Laboratory Medicine, Emory University, Atlanta, GA30322; †Department of Pediatrics, Aflac Cancer and Blood Disorders Center, EmoryUniversity School of Medicine, Atlanta, GA 30032; and ‡Puget Sound Blood CenterResearch Institute, Seattle, WA 98102

1S.R.S. and J.S.L. contributed equally to this work.

Received for publication April 3, 2013. Accepted for publication September 6, 2013.

This work was supported by discretionary funds provided by Emory University andthe Puget Sound Blood Center (to J.C.Z.).

Address correspondence and reprint requests to Dr. James C. Zimring, Puget SoundBlood Center Research Institute, 1551 Eastlake Avenue E, Seattle, WA 98102. E-mailaddress: [email protected]

The online version of this article contains supplemental material.

Abbreviations used in this article: DiD, 1,19-dioctadecyl-3,3,39,39-tetramethylindodi-carbocyanine perchlorate; DiI, 1,19-dioctadecyl-3,3,3939-tetramethylindocarbocyanineperchlorate; DiO, 3,39-dihexadecyloxacarbocyanine perchlorate; GPA, glycophorin A;HOD, hen egg lysozyme–OVA–Duffy; KO, knockout; WT, wild-type.

Copyright� 2013 by The American Association of Immunologists, Inc. 0022-1767/13/$16.00

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induced alteration of recognized Ag as a mechanism to decreasesurface-bound Abs.Ag modulation has been difficult to study in vivo, because the

target cells typically have ongoing synthesis of the target Ag, re-sulting in steady state equilibria that complicate analysis of thefate of particular Ag molecules (21, 25). Moreover, as target cellshave ongoing division, tracking their numerical destruction overtime (or lack thereof) after Ab binding is likewise challenging (21,25). To circumvent both of these limitations, we have made specificuse of a model of Ag modulation using RBCs as cellular targets.Mature RBCs are postmitotic and lack ongoing protein synthesis(26). It is for this reason that transfer of a distinct RBC populationinto an Ag2 recipient allows examination of specific consequences,including cellular clearance and the potential development ofprotection against Ab-mediated removal, without the confound-ing variables of cell division or synthesis of additional protein. Inthis study, we use a recently described RBC Ag–Ab system usingRBCs that express a unique model Ag: the hen egg lysozyme(HEL)–OVA–Duffy (HOD) Ag (27, 28). Using this system, wedescribe RBCs escaping destruction by Abs through Ag modula-tion. More importantly, the clearance mechanisms require FcgRs,but not complement. This feature allows a mechanistic focus onAg modulation and its effects in an isolated FcgR-dependent path-way. We report that in addition to mediated clearance, FcgRs arethemselves instrumental in bringing about Ag modulation of Abtargets. Taken together, these results elucidate details of a poorlyunderstood mechanism of cellular alteration in response to Abbinding that may lead to protection against FcgR-mediated effectorresponses in vivo.

Materials and MethodsMice

C57BL/6 (B6), FVB, and C3 knockout (KO) mice were purchased fromThe Jackson Laboratory (Bar Harbor, ME). FcgR KO mice (Fcer1g) werepurchased from Taconic Farms All mice were used at 8 to 14 wk of age.HOD mice consisted of transgenic animals (FVB background) expressinga fusion protein HEL, Ova, and Duffy b (also known as HOD mice) (27).All breeding (including FcgR KO and C3 KO mice) was performed by theEmory University Department of Animal Resources Husbandry Services,and all procedures were performed according to approved InstitutionalAnimal Care and Use Committee protocols.

Abs and passive immunization

The mouse anti-Fy3 (anti-HOD, MIMA29, IgG2a) and mouse anti-M(anti-GPA, 6A7, IgG1) were a generous gift from Marion Reid and GregHalverson at the New York Blood Center. Abs were purified by protein Gchromatography (Bio X Cell, West Lebanon, NH). The wild-type (WT)C57BL/6 mice were passively immunized with 500 ml 200 mg MIMA29 inPBS or 25 mg 6A7 in PBS, or were given PBS alone by tail-vein injection2–5 h prior to transfusion.

Fluorescent labeling and transfusion of murine RBCs

HOD transgenic mice, HOD X GPA transgenic mice, and WT FVB micewere anesthetized with isoflurane and exsanguinated by enucleation. Bloodcollected was washed with PBS and labeled with one of three differentlipophilic dyes used to track the survival of these RBCs posttransfusion, asoutlined previously (29, 30). Briefly, HOD blood was typically labeled with1,19-dioctadecyl-3,3,3939-tetramethylindocarbocyanine perchlorate (DiI), andFVB blood was typically labeled with 3,39-dihexadecyloxacarbocyanineperchlorate (DiO). After labeling, HOD and WT FVB blood cells weremixed at a 1:1 ratio and brought to a 20% hematocrit with LPS-free PBS.Each recipient was transfused by tail-vein injection, with 500 ml of theabove prepared blood mixture. For immunized mice receiving a secondtransfusion, recipients first received unlabeled HOD blood followed bya mixture of DiI-labeled HOD blood and DiO-labeled FVB blood 2 d later.

At 2 d posttransfusion, the mice were exsanguinated by enucleation, andblood was mixed with freshly collected HOD blood labeled with a differentlipophilic dye, typically 1,19-dioctadecyl-3,3,39,39-tetramethylindodicarbocya-nine perchlorate (DiD). The fresh DiD-labeled HOD blood (“fresh HOD

blood”) was mixed with blood collected from recipients of the first transfusionat a 1:1 ratio with the surviving DiI-labeled HOD RBCs (“experienced HODblood”). The total mixture was brought to a 20% hematocrit with LPS-freePBS, and 500 ml was transfused by tail-vein injection into fresh C57BL/6mice.

RBC survival

After both the first and second transfusions, mice were anesthetized, and25–50 ml blood was collected retro-orbitally at 2 h, 20 h, and 2 d post-transfusion. After the first transfusion, samples were analyzed by flowcytometry, from which a ratio of DiI-labeled HOD blood to DiO-labeledFVB blood within each animal was calculated. These ratios from Ab-treated animals were then normalized to PBS-treated animals and graphedas percent survival. After the second transfusion, the ratio of DiI-labeled“experienced” HOD blood cells to DiO-labeled FVB blood cells wascalculated, and the resulting ratios were normalized to the ratios calculatedfrom RBCs transfused into two consecutive PBS-treated mice. Similarly,the ratio of DiD-labeled “fresh” HOD RBCs (never previously transfused)to DiO-labeled FVB RBCs was calculated. However, these DiD-HOD toDiO-FVB RBC ratios were each normalized to their PBS counterpartstransfused with the same blood mixture (both originated from the samefirst transfusion recipients). Survival graphs were all calculated and createdwith Prism software. Error bars represent SD.

RBC characterization

A direct antiglobulin test was performed on samples collected after first andsecond transfusions at a 1:100 dilution of goat anti-mouse Ig Abs (BDBiosciences) or anti-C3 (Cedarlane). The samples were also stained witha primary Ab, MIMA29, an anti-Fy6 Ab, or an anti-GPA Ab at a con-centration of 1 mg/100 ml in FACS buffer (2% BSA, 0.9% EDTA in PBS),then with a secondary Ab, goat anti-mouse Igs (BD Biosciences Phar-mingen), diluted 1:100 in FACS buffer, as outlined previously (31). Thesamples were also stained with Rat anti-TER119 (BD Biosciences Phar-mingen), also diluted to 1:100 with FACS buffer; Rat IgG2b k (BD Bio-sciences Pharmingen) Abs were used as an isotype-matched control.

ResultsGeneration of an experimental model to Ag modulation asa result of Ab engagement

Our experimental system takes advantage of a transgenic mousethat expresses a chimeric model Ag on RBCs, consisting of a fusionof HEL to the C-terminal half of OVA to the human Duffy Bglycoprotein. The HOD Ag provides several well-defined immu-nological epitopes on the same protein, allowing a detailed studyof changes to an RBC surface protein during binding of differentAbs (see Fig. 1A for diagram of HOD Ag). To facilitate visualiza-tion and examination of HOD (Ag+) cells following adoptive trans-fer into HOD2 (Ag2) recipients, we labeled HOD (Ag+) cells withthe fluorescent dye DiI, to enable direct flow cytometric exami-nation of Ag+ cells following Ab engagement in vivo (Fig. 1A). Todetermine whether potential cellular alterations specifically oc-curred on Ag+ cells within a given recipient, a second populationof Ag2 (Ag2) cells was labeled with a different fluorescent dye,DiO, to serve as an Ag2 control (Fig. 1A).Adoptive transfer of either DiI- or DiO-labeled RBCs, followed

by evaluation of recipient peripheral blood by flow cytometry,demonstrated that single labeling resulted in a distinct and de-tectable population for either DiO (Fig. 1B) or DiI (Fig. 1C).Moreover, neither DiO nor DiI cells cross-fluoresced on thechannel of the other dye. Staining with anti-TER119, an RBC-specific Ag, confirmed that essentially 100% of events wereTER119+ after gating on DiI or DiO populations (Fig. 1B, 1C).Finally, DiI-labeled Ag+ RBCs and DiO-labeled Ag2 RBCs weremixed prior to adoptive transfer, and peripheral blood of recipientswas analyzed. Not only were the DiO+ and DiI+ populations easilydistinguishable, but the Ag+ HOD RBCs (DiI+) stained positivewith an Ab to HOD (anti-Fy3), whereas no staining was observedon WT Ag2 RBCs (Fig. 1D). Thus, in aggregate, this techniqueallows the introduction of RBCs into a recipient that has Abs

5014 Ag MODULATION PROTECTS RBCs FROM Abs

against a defined Ag, combined with the ability to simultaneouslyevaluate RBC survival and to visualize and characterize alterationsto the targeted Ag. Owing to the nature of RBCs, neither cellulardivision nor new Ag synthesis is able to confound interpretation ofcellular survival or Ag modulation.

In vivo binding of Ab to RBC Ag and the effects on RBCsurvival

To study the effects of Ab engagement on RBC survival, a mixureof Ag2 (DiO) and Ag+ (DiI) RBCs was transferred i.v. into micethat had been passively immunized with a mouse IgG2a mAbagainst one epitope on the HOD Ag (anti-Fy3). In any given re-cipient, survival of Ag+ RBCs was normalized to survival of Ag2

RBCs, to allow an internal control for any variability in adoptivetransfer or bloodletting. Control animals that were not immunized(PBS injected) were used to establish the baseline survival of Ag+

RBCs in the absence of Ab binding. To evaluate binding of anti-Fy3 to Ag+ RBCs, peripheral blood of recipients was stained withanti-Ig and evaluated by flow cytometry. Anti-Ig was nonreactivewith either Ag2 or Ag+ RBCs in control mice injected with PBS(Fig. 1E). In contrast, Ag+ RBCs in immunized mice were stronglyreactive with anti-Ig, whereas no signal was detected on Ag2 RBCs(Fig. 1F). Together, these data demonstrate selective in vivo bindingof anti-Fy3 to Ag+ RBCs expressing the HOD Ag.Analysis of RBC survival over time indicated that Ag+ RBCs

had a dramatically decreased survival compared with survival in

FIGURE 1. Kinetics of RBC clearance after transfer into immunized recipients. (A) Schematic representation of the HOD Ag on Ag+ cells. Ag+ or Ag2

cells are labeled with lipophilic dyes, DiI or DiO, respectively. Following labeling, Ag+ and Ag2 cells are mixed at a defined ratio and transferred into

immunized or nonimmunized recipients. At the indicated time points posttransfer, peripheral blood is obtained, and labeled RBCs are evaluated for vi-

ability, Ab binding, and potential changes to Ag+ cells. (B and C) Flow cytometric gating strategy to examine labeling efficiency prior to transfer for Ag+

and Ag2 cells. (D) Examination of cells following transfer in vivo into nonimmunized recipients for the presence or absence of Ag. (E and F) Peripheral

blood was obtained at the times indicated posttransfer from nonimmunized (E) or immunized (F) WT recipients. Flow cytometry was used to evaluate

survival and surface Ab on Ag+ cells (solid black line) or Ag2 cells (gray) 10 min posttransfer. (G) Quantification of Ag+ cell clearance following transfer

into nonimmunized or immunized WT recipients. Representative flow plots are reflective of three independent experiments. Each experiment represents

three to five mice per group at each time point analyzed.

The Journal of Immunology 5015

Ag2 RBCs in mice that received anti-Fy3 but had a normal sur-vival in control animals that received PBS (Fig. 1G). However,clearance kinetics were neither linear nor uniform. Although nearly40% of Ag+ cells cleared within the first 2 h following adoptivetransfer, 2 d were required for an additional 40% to clear (Fig. 1G).These clearance kinetics indicate an alteration to the biology re-garding RBC clearance after 2 h.

RBCs surviving despite Ab binding are resistant to clearance

The existence of a rapid phase of RBC clearance followed bysignificantly slower clearance raised the hypothesis that a sub-population of RBCs may resist clearance mechanisms. However,these findings were also compatible with alternative and moretrivial hypotheses, in particular, depletion of Ab or saturation ofRBC clearance mechanisms. To test these hypotheses, two separateapproaches were taken. In the first approach, a two-stage transferexperiment was carried out (see Fig. 2A for diagram). In the firsttransfer, a mixture of Ag+ (DiI) and Ag2 (DiO) RBCs was trans-ferred into animals immunized with anti-Fy3 (as above), andthe rapid phase of RBC clearance was allowed to occur. At2 d later, the RBCs that had survived were recovered by exsan-guinating recipient mice. The recovered RBCs were then trans-ferred to new mice (second transfer), which had been passivelyimmunized with anti-Fy3. In this way, surviving RBCs were ex-posed to a fresh dose of Ab and RBC clearance machinery thathad not yet consumed Ab-bound RBCs. The above hypothesespredict that if the slowed clearance after 40% removal was due toAb depletion or saturation of clearance mechanisms, then a new“rapid phase” should be observed during the second transfer. Incontrast, if the RBCs have a phenotype resistant to clearance, thenin the second transfer, they should have kinetics similar to those ofthe slow clearance phase.During the first transfer, and consistent with results in Fig. 1,

Ag+ RBCs had a rapid clearance phase followed by a slower clear-ance phase (Fig. 2B). However, only a small and limited rapidclearance phase was observed during the second transfer (Fig. 2C).

Rather, the majority of Ag+ RBCs cleared at the slower rate, con-sistent with the second phase of clearance (see Fig. 1). This resultwas not due to simply transfusing a smaller number of RBCs asa result of the rapid clearance phase eliminating 40% of RBCs, asdecreasing the RBC number of Ag+ RBCs during the first transferhad the opposite effect (Supplemental Fig. 1). It was not possiblein the second transfer to have a control group that had no Ab bound,as the Ab from the first transfer remains bound when put into asecond mouse (Supplemental Fig. 1). However, we did control forthe possibility that slower clearance was due to just having circu-lated in a mouse (See Fig. 2A, group 2), which did not prevent therapid clearance phase in the second transfer but instead resulted inenhanced clearance (Fig. 2D).Although these results suggested that Ag+ cells may acquire

resistance to Ab-mediated removal, this approach did not specif-ically determine whether immunized recipients retain the capacityto clear Ag+ cells following the development of reduced rates ofclearance. To test this, Ag+ cells were transferred into immunizedrecipients, followed by a second transfer of Ag+ cells into thesame recipients 2 d later. As a control, immunized recipients notpreviously exposed to Ag+ cells were transferred with Ag+ cells inparallel (Fig. 3A). Ag+ cells experienced similar levels of clear-ance in immunized recipients that previously received Ag+ cellsas immunized recipients not previously exposed to Ag+ cells,strongly suggesting that immunized recipients retain the ability toremove Ag+ cells following the development of reduced rates ofclearance (Fig. 3B, 3C). As a whole, our interpretation of thesedata is that Ab-bound HOD Ag+ RBCs that survive the rapidclearance phase after initial transfer are resistant to subsequentclearance.

Clearance of HOD RBCs by anti-Fy3 requires FcgRs, butnot C3

To further investigate mechanisms of clearance resistance, it wasnecessary to establish the fundamental mechanisms of clearanceitself. The two main pathways involved in clearance of Ab-coated

FIGURE 2. RBCs surviving the rapid clearance phase have a resistance to clearance by anti-Fy3. (A) Diagram of experimental design involving an initial

transfer with clearance, recovery of transferred RBCs, and then a second transfer into a fresh recipient. (B) Kinetics of clearance during first transfer. (C)

Kinetics of clearance during second transfer following first transfer into immunized recipients (Group 1). (D) Kinetics of clearance during second transfer

following first transfer into nonimmunized recipients (Group 2). Data are representative of at least three independent experiments, and each experiment

represents three to six mice per group at each time point analyzed.


RBCs in vivo involve either FcgRs or complement. However,alternative pathways have also been described that do not requireeither FcgRs or complement (28). Thus, to evaluate clearancemechanisms of anti-Fy3 and HOD, transfer experiments werecarried out in mice with targeted deletions of either the C3 com-ponent of complement or the common g-chain of FcgRs.C3 is the main complement opsonin that causes RBC con-

sumption and is also instrumental to downstream formation of themembrane attack complex. Clearance in C3 KOmice had a kineticsand magnitude similar to that observed in WT recipients (Fig. 4A).To test whether C3 is deposited on Ag+ RBCs after anti-Fy3binding, peripheral blood samples were obtained at 2 h post-transfusion from WT recipients and were stained with anti-C3.Anti-C3 reactivity was detected in the presence of anti-Fy3 ina population of Ag+ (DiI) RBCs (Fig. 4B). This C3 depositionrequired the presence of the target Ag, as no C3 was detected onAg2 RBCs (DiO) (Fig. 4B). The deposition of C3 on HOD RBCs

was not a spontaneous event or a function of the nonclassicalpathway, as no C3 was detected on either Ag+ or Ag2 RBCs incontrol mice that received PBS (Fig. 4B). Thus, deposition of C3in this system requires that Ag and Ab be simultaneously present.Representative flow plots during clearance in the C3 KO mice areshown and, as predicted owing to the targeted deletion of C3, noanti-C3 reactivity is observed in either group (Fig. 4C). Together,these data indicate that C3 is indeed deposited on the surface ofsome HOD RBCs after binding of anti-Fy3; however, the func-tional effect of C3 binding is unclear with regard to clearance, asclearance curves are normal in the C3 KO animal. Thus, these datado not rule out the involvement of C3 but do demonstrate that C3is not required for clearance in this system.Although C3 engagement is thought to be the primary com-

plement effector pathway whereby Abs initiate complement, sev-eral C3-independent pathways of complement activation havebeen described (32), suggesting that downstream targets of com-

FIGURE 3. Immunized recipients maintain the ability to clear Ag+ cells. (A) Diagram of experimental design involving only a single transfer into

immunized recipients (Group 1) or examination of clearance in immunized recipients who had received Ag+ cells prior to the second Ag+ cell transfer. (B

and C) Kinetics of clearance of Ag+ cells following the first transfer (B) or subsequent transfer into recipients that had already been immunized and received

Ag+ cells (C). Data are representative of at least three independent experiments, and each experiment represents three to five mice per group at each time

point analyzed.

FIGURE 4. Clearance of HOD RBCs by anti-Fy3 requires FcgRs, but not C3. (A) Clearance kinetics of Ag+ RBCs in C3 KO mice immunized with anti-

Fy3 or saline. (B) Flow cytometric examination of Ag+ cells (solid black line) or Ag2 cells (gray) for surface C3 binding following transfer into WT

recipients. (C) Representative DiI 3 DiO plots for calculating RBC survival and flow cytometric examination of Ag+ cells (solid black line) or Ag2 cells

(gray) for surface C3 binding following transfer into C3 KO recipients. (D) Clearance kinetics of Ag+ RBCs in FcgR KO mice immunized with anti-Fy3 or

saline. Representative flow plots are reflective of three independent experiments. Each experiment represents three to five mice per group at each time point

analyzed.


plement activation may be involved in cellular clearance. To testthis, we next transferred Ag+ cells into immunized FVB recipi-ents, which are deficient in the C5 component of complementrequired for initiation of the membrane attack complex (33). Im-munized FVB recipients displayed a similar ability to clear Ag+

cells (Supplemental Fig. 2), strongly suggesting that Ag+ cellclearance occurs independent of the downstream complementeffector molecules. Thus, these data do not rule out the involve-ment of C3 or C5 but do demonstrate that C3 and C5 are notindividually required for clearance in this system.To determine the role of FcgRs in this process, the same ex-

periment was carried out in mice with a targeted deletion of thecommon Fc g-chain, resulting in a lack of functional FcgRI,FcgRIII, and FcgRIV (16). No clearance of Ag+ cells was ob-served following transfer into immunized FcgR KO recipients(Fig. 4D), indicating that FcgRs are required for RBC clearance inthis system. Because essentially no clearance was observed in theFcgR KO mice, this also suggests that complement is not suffi-cient to induce RBC clearance. Together, these data indicate thatFcgRs are required for clearance of HOD RBCs by anti-Fy3 andthat C3 is neither required nor sufficient for clearance.

RBCs resistant to clearance undergo Ag modulation of surfaceHOD that correlates with resistance to clearance

To further characterize Ag–Ab interactions on the RBCs, we di-rectly examined Ag+ cells for Ab engagement at various pointsfollowing adoptive transfer into immunized recipients. Peripheralblood was collected at the indicated time points, stained with anti-Ig, and analyzed by flow cytometry (Fig. 5A). Although the levelof bound Ab remained high during the initial rapid phase ofclearance, the amount of detectable Ab on Ag+ cells progressivelydecreased over time (Fig. 5A). The staining was specific for thepresence of anti-Fy3, as no signal was detected on Ag2 RBCs(Fig. 5A) or on either Ag+ or Ag2 RBCs in control mice thatreceived PBS (Fig. 5A and Supplemental Fig. 3).As immunization of recipients reflected passive transfer of Ab,

it remained possible that reduced Ab engagement simply reflectedrapid consumption of passively administered Ab. However, stainingof recovered RBCs with anti-Fy3 itself, followed by anti-Ig, failedto increase the signal, indicating that the available anti-Fy3 bindingsites were saturated (Fig. 5B). In contrast to the observed decreasein Ag seen on Ag+ RBCs in anti-Fy3–immunized mice, Ag+ RBCsrecovered after transfer into PBS-treated mice retained full staining(Fig. 5B, Supplemental Fig. 3). Apparent loss of detectable Ag didnot appear to reflect wholesale loss of the plasma membrane, ascells experiencing Ag modulation isolated from immunized recip-ients displayed forward scatter profiles very similar to those ofAg+ cells transferred into nonimmunized controls (Fig. 5C, 5D).Together, these data indicate that the amount of detectable Ag onthe transferred Ag+ RBCs decreases as a function of being trans-ferred into an animal with circulating anti-Fy3, and establishes thephenomenon of Ag modulation in this system.

Effect of Ab density on RBC Ag decrease and clearance

The above data indicate that the decrease in levels of Ab en-gagement on Ag+ cells paralleled the decreased kinetics of RBCclearance and the generation of RBC resistance to clearance (seeFigs. 1G, 2). These observations led us to hypothesize that alter-ations in surface RBC Ag, and thus decreased Ab engagement,was involved with decreased RBC clearance. However, it is im-portant to note that although Ag modulation decreases detectableAg, Ag-bound Ab remains readily detectable. Thus, for Ag mod-ulation to have an effect on RBC clearance, there would haveto be a density dependence of FcgR ligation. To test this hy-

pothesis, we incubated cells with varying levels of Ab or no Abin vitro prior to adoptive transfer in an effort to recapitulate thepotential impact of reduced Ab levels on Ag+ cell removal (Fig.6A). Incubation of Ag+ cells with Ab in vitro enabled isolation ofRBCs with Ag saturation [hi], an Ab intermediate population [int],or cells with no Ab [0] prior to transfer (Fig. 6B, SupplementalFig. 3). Each population of RBCs was then transferred into WTmice (no passive Ab into the mice), and mice were bled at theindicated time. The amount of surface IgG was determined bystaining with anti-Ig, whereas the amount of Fy3 Ag was deter-mined by staining with anti-Fy3 followed by anti-Ig.Compared with control HOD RBCs that were transferred with-

out preincubation with Ab (Fig. 6C, top row), transfer of Ab-saturated cells [hi] displayed significant decrease in bound Abover time (Fig. 6C, bottom row). Likewise, staining with anti-Fy3followed by anti-Ig demonstrated decreased detectable Ag in [hi]RBCs (Fig. 6D, bottom row). This observed pattern in [hi] RBCswas similar to what was observed following adoptive transfer ofAg+ cells into immunized recipients (see Fig. 5). In contrast, [int]cells failed to display a similar decrease in surface Ab from pre-transfer levels (Fig. 6C, middle row). Moreover, staining with anti-Fy3 and anti-Ig increased the signal on [int] cells to a level similarto that in control HOD RBCs that were transferred without pre-incubation with Ab (Fig. 6D, middle and top rows; SupplementalFig. 3). Thus high levels of Ab, but not intermediate levels, resultin Ag modulation over time.To test the effect of [0], [int], and [hi] levels of surface RBC Ab on

RBC survival after transfer, circulating Ag+ RBCs were enumeratedby the same DiI/DiO techniques used above. Whereas [hi] levels ofAb caused rapid clearance, [int] levels of Ab caused substantiallyslower clearance (Fig. 6E). The [hi] and [int] Ab incubations weretitrated to mimic Ab levels on Ag+ RBCs prior to transfer and afterthe rapid clearance phase, respectively. Thus, these findings areconsistent with Ag modulation, resulting in a sufficient decrease insurface Ab to render RBCs resistant to clearance. Together, thesedata indicate that both clearance and Ag modulation are a functionof the amount of RBC surface-bound Ab.

FcgRs, but not C3, is required for decrease in detectable Ag

Although C3 was neither required nor necessary for clearanceof transferred HOD RBCs, this does not exclude its possible rolein Ag modulation. Indeed, Taylor et al. (34) have reported that C3deposition on Ig itself can prevent binding of secondary Abs throughdirect blocking or steric hindrance. To test this hypothesis, Agmodulation was evaluated after transfer into C3 KO mice. Transferof Ag+ cells into immunized C3 KO mice resulted in a decrease indetectable Ag similar to that in WT recipients (Fig. 7A, 7B).Similar results were obtained following transfer of Ag+ cells intoC5-deficient FVB immunized recipients (Supplemental Fig. 4).These data indicate that C3 and C5 are not required for Ag mod-ulation in this system, and we thus reject the hypothesis that themechanism of Ag decrease is masking by C3 or C5 deposition.To test the requirement for FcgR for Ag modulation, a mixture

of Ag+ and Ag2 cells was transferred into FcgR KO immunizedrecipients. In the absence of FcgRs, Ab persisted on cells unal-tered over extended periods (Fig. 7C). As above, RBCs did notclear in FcgR mice that were immunized with anti-Fy3, whereasnormal clearance occurred in WT animals (Fig. 7D–F). These dataindicate that FcgRs are required for the process of Ag modulation.

Decrease in detectable Ag extends intra- but notintermolecularly

To determine whether Ag modulation was specific to the epitoperecognized by anti-Fy3, we compared levels of Fy3 with an epitope


on the opposite end of HOD (Fy6; see Fig. 8A). Ag+ cells wereisolated at various time points following transfer and were incu-bated with an anti-Fy6 Ab, followed by detection of total Abbound to cells, similar to the method used to determine whetheralterations in the Fy3 target epitope occurred over time. Althoughthe Fy6 epitope remained detectable, with unaltered levels of Agon cells transferred into nonimmunized recipients, the Fy6 epitopeexperienced a decrease similar to that observed for the Fy3 epi-tope over time in immunized mice (Fig. 8B–D). These data indi-cate that a separate epitope on the same molecule, and not recognized

by anti-Fy3, undergoes a simultaneous and similar decrease in de-tectability.To test whether Ag modulation was generalized to epitopes on

separate molecules, two approaches were taken. In the first ap-proach, we tested the expression of Ter119, an RBC-specific Agunrelated to HOD. Ter119 RBCAg expression remained unchangedover the same time and under the same conditions (Fig. 8E). In thesecond approach, we crossed HOD transgenics by transgenicsexpressing the human glycophorin A (GPA) Ag, a human Ag similarto the mouse Ter119 Ag, on RBCs and transferred these double

FIGURE 5. Ag-positive cells undergo Ag modulation coincident with development of resistance to clearance. (A) Flow cytometric examination of Ag+

cells (solid black line) or Ag2 cells (gray) for bound Ab following transfer into immunized or nonimmunized recipients at the times indicated. (B) Flow

cytometric examination of Ag+ cells (solid black line) or Ag2 cells (gray) for detectable Ag following transfer into immunized or nonimmunized recipients

at the time points indicated. (C) Examination of forward scatter as a function of bound Ab at the time points indicated. (D) Overlay of the forward scatter

profile of Ag+ cells isolated for nonimmunized (gray) or immunized (solid black line) at the time points indicated. Representative flow plots are reflective of

three independent experiments. Each experiment represents three to five mice per group at each time point analyzed.


positive cells into nonimmunized, anti-Fy3–immunized, or anti-GPA–immunized recipients. Although nonimmunized recipientsfailed to clear double Ag+ cells, anti-Fy3–immunized and anti-GPA–immunized recipients each effectively cleared double positivecells (Fig. 9A), consistent with previous results and those of thisstudy (35). Examination of bound Ab and detectable Ag demon-strated that Ab could be detected only on double Ag+ cells followingtransfer into immunized recipients and that double positive RBCsexperienced modulation of HOD Ag, but not GPA Ag, followingtransfer into anti-Fy3–immunized recipients (Fig. 9B). Conversely,transfer of double Ag+ cells into anti-GPA–immunized recipientslikewise resulted in Ag modulation of the GPA Ag, whereas the

HODAg remained unaltered (Fig. 9B). Together, these data indicatethat a decrease in detectable Ag appears to reflect specific modu-lation of the target Ag following Ab engagement.

DiscussionInnate immunity and adaptive immunity have evolved multipleeffector pathways to protect the host from pathological microbes.Abs represent one such pathway and have the capacity to destroythe targets they bind through both fixation of complement andligation of FcgRs. However, the presence of such Abs necessitatesan intrinsic risk of damaging self-tissues. As an evolutionary re-sponse to this damage, multiple pathways have been described

FIGURE 6. Cells with intermediate levels of Ab exhibit reduced sensitivity to clearance. (A) Schematic representation of the experimental design. (B)

Distinct populations of Aghigh or Agintermediate cells transferred into nonimmunized recipients. (C) Examination of Ab bound to transferred cells at the

indicated time points on Ag+ cells (solid black line) or Ag2 cells (gray). (D) Examination of levels of detectable Ag on Ag+ cells (solid black line) or Ag2

cells (gray) at the indicated time points. (E) Clearance kinetics of RBCs pretreated with [0], [int], or [hi] concentrations of anti-Fy3, followed by transfer

into WT recipients. Representative flow plots are reflective of three independent experiments. Each experiment represents three to five mice per group at

each time point analyzed.


by which self-tissues can inactivate complement, thus mitigatingdamage from autoantibodies or alloantibodies. In contrast, littlehas been described regarding FcgR-mediated pathways and theextent to which protection pathways have evolved. One pathwaythat has been put forward is referred to in this article as “Agmodulation,” a process by which target Ags change so as to de-crease Ab binding and thus limit FcgR ligation. Detailed analysisof the effects of Ag modulation requires a system in which FcgRsare isolated as an Ab effector pathway (i.e., complement does notplay a role). Moreover, one must control issues of cellular pro-liferation and Ag resynthesis by target cells during any ongoingAg modulation. In this article, we describe just such a system bytaking advantage of RBCs, which neither proliferate nor synthe-size protein and are cleared by an FcgR dependent/complement-independent pathway.The data presented generate novel understanding of Ag mod-

ulation at several different points. First, the presence of bothanti-Fy3 and FcgRs is required for both RBC clearance and Ag

modulation. Hence, the biology and the multiple effects of Ab-induced FcgR ligation overlap. One trivial explanation that mightcome from these observations would be that RBCs with a baselinelevel of higher Ag expression are cleared, whereas those with abaseline lower Ag expression are not. Thus, what appears to beongoing Ag modulation on RBCs actually just represents selectiveremoval of a population with high Ag expression. However, wereject this interpretation as mathematically inconsistent withstaining of RBCs at baseline. After 2 h, 50% of the Ag+ RBCs arestill circulating and have a 5-fold decrease in surface Ag. How-ever, prior to transfer into immunized animals, only a very smallpercentage of RBCs have lower baseline levels of Ag on thesurface (see Fig. 1). Accordingly, our interpretation is that sur-viving RBCs started with high levels of detectable Ag and un-derwent an Ag modulation process that substantially decreaseddetectable Ag.In the above context, the simultaneous requirement for FcgR

to carry out both clearance and Ag modulation is a unique and

FIGURE 7. Fcg receptor–mediated Ab-induced Ag modulation. (A–C) Examination of bound Ab or detectable Ag 2 d following transfer of Ag+ cells

(solid black line) or Ag2 cells (gray) into nonimmunized or immunized WT (A), C3 KO (B), or FcgR KO (C) recipients, as indicated. (D) Examination of

Ag+ cell clearance in nonimmunized or immunized FcgR KO recipients, as indicated. (E and F) Quantification of cellular clearance 2 d following transfer of

Ag+ cells into nonimmunized or immunized WT (E) or FcgR KO (F) recipients, as indicated. Representative flow plots are reflective of three independent

experiments. Each experiment represents three to five mice per group at each time point analyzed.


novel finding. This indicates an FcgR-dependent biology thatresults in the modulation of presynthesized Ag on the RBC sur-face. The precise details of the mechanism by which this occursare not clear at all levels; however, we can make some distinctconclusions based upon the current data. First, according to pub-lished literature, C3 that deposits on cell-bound Ab can modify itsuch that anti-Ig no longer recognizes the Ab (34, 36, 37). Giventhat C3 deposition was detected after anti-Fy3 bound to Ag+ RBCs(see Fig. 3), and given the known phenomenon, this seemeda likely hypothesis. However, we reject this hypothesis based uponthe fact that Ag modulation occurs unabated in C3 KO mice.Similar results were observed following transfer into immunizedFVB C5-deficient recipients (33). This observation does not ad-dress the more general issue of any modification of anti-Fy3 (otherthan involving C3 or C5) that may render it undetectable by anti-Ig. However, the observation that both Fy6 and Fy3 undergo Agmodulation after exposure to anti-Fy3 provides additional evi-dence that simple modification of the anti-Fy3 is not a likelymechanism. Together, these data indicate that the mechanism ofAg modulation is not modification of the Ag-bound Ab, and likelyrepresents alteration to the Ag itself.The exact fate of the HOD Ag, after binding of anti-Fy3, is not

clear; indeed, possibilities include complete removal from thecellular membrane, partial removal of select epitopes, or proteinmodification so as to render the Ag undetectable by Ab. Themodulation of Fy6 by anti-Fy3 rejects the hypothesis that anti-Fy3is causing an epitope-selective destruction of the protein. Moreover,reduction of the protein and chemical denaturation seem unlikelymechanisms, as both anti-Fy3 and anti-Fy6 recognize linear peptidesequences. In addition, the selective modulation of the Fy3 Ag fol-lowing transfusion of HOD X GPA RBCs also suggests that anti-Fy3

engagement results in specific alterations to the target Ag. Althoughcomplete removal of a transmembrane protein from the RBC surface,without damaging the RBC, may seem unlikely, at least one humancase of Ag modulation during autoimmune hemolytic anemia pro-vided substantial evidence that the Ag was removed from the RBC(38). This removal would not necessarily require pulling the pro-tein out of the membrane, which seems thermodynamically unlikely.Rather, it may involve trogocytosis of aggregated Ag complexes,which has been observed in the context of Ag modulation of CD20by chronic lymphocytic leukemia in response to mAb immuno-therapy (i.e., rituximab) (25). As some cases of autoimmune he-molytic anemia, in which changes in cell size may be detectable,reflect Ab engagement of high-density Ags (39), the lack of de-tectable alterations in cell size may simply reflect lower Ag densityin the present system with a corresponding reduction in the amountof membrane removed. As a result, subtle changes in Ag levels, asobserved following Ag modulation, may not produce enough lossof membrane to be detected with the tools used in this study. As aresult, we propose a model by which Ab binding Ag and ligatingFcgR results in removal of the Ag from the membrane.The process of Ag modulation has been observed in numerous

settings, and strong human data indicate that Ag modulation occursby several different mechanisms, depending upon the nature of theAb, the Ag, and the target cell. Very much as in humans, the samediversity of mechanisms appears to be present in mice. We havepreviously reported that Ag modulation of murine HEL on RBCsrequires the simultaneous binding of two separate Abs that bind todistinct epitopes. However, Abs that engage HEL in this system failto induce RBC clearance, making it difficult to determine whetherAg modulation can protect cells from Abs capable of inducing RBCremoval. However, such appears not to be the case in the current

FIGURE 8. Anti-Fy3 induces coincident Ag modulation of both Fy3 and a third party Ag, Fy6. (A) Schematic of the HOD Ag. (B and C) Examination of

detectable Ag for Fy6 (B) or Fy3 (C) on Ag+ cells (solid black line) or Ag2 cells (gray) at the time points indicated following transfer into nonimmunized or

immunized recipients. (D) Examination of Ab on Ag+ cells (solid black line) or Ag2 cells (gray) at the time points indicated following transfer into

nonimmunized or immunized recipients. (E) Examination of cell surface Ter119 staining on Ag+ cells (solid black line) or Ag2 cells (gray) 5 d following

transfer into immunized or nonimmunized recipients, as indicated. Ag+ cells (black dotted line) or Ag2 cells (light gray) stained with isotype control are

also shown. Representative flow plots are reflective of three independent experiments. Each experiment represents three to five mice per group at each time

point analyzed.


setting, as a monoclonal anti-Fy3 resulted in Ag modulation andclearance. Similarly, the ability of anti-GPA to induce Ag modu-lation demonstrates that Ag modulation is not limited to anti-Fy3.The unique biphasic clearance pattern induced by anti-GPA isconsistent with previous studies (35). Given the potential in-volvement of complement in anti-GPA–mediated clearance (40),complement receptors may initially trap RBCs following Ab-mediated complement deposition and then release some RBCsfollowing subsequent complement degradation, as suggested byprevious studies examining the consequences of complement de-position on RBCs (36, 41). Although additional studies are neededto further understand the mechanisms of anti-GPA–induced clear-ance and Ag modulation, these results suggest that alterations inAg following Ab engagement may reflect a general mechanismof cellular protection from Ab-mediated removal. Thus, althoughthe current findings cannot be used to draw generalized con-clusions regarding Ag modulation, Ag modulation appears to re-flect a general phenomenological descriptor that captures severaldifferent processes with a common outcome. However, to the bestof our knowledge, the current report represents the first descrip-tion of a new pathway of Ag modulation, in which FcgRs are re-quired both for clearance and for the process of Ag modulation itself.Although Ag modulation and clearance appear to use the same

FcgR system, whether the same FcgRs mediate clearance and Agmodulation remains unknown. Previous studies suggest that IgG2aAbs preferentially engage FcgRIV, whereas IgG1 and IgG3 preferto bind FcgRIII and FcgRI, respectively (42, 43). As FcgR KOrecipients were used in the current study, it remains possible thatthe IgG2a anti-Fy3 used in this study preferentially triggersFcgRIVs following engagement of Ag+ cells in vivo. In contrast,class switch variants of anti-Fy3 may preferentially engage al-ternative FcgRs. As it remains possible that the FcgRs responsiblefor clearance may be different from those that induce Ag modu-

lation, manipulation of Ab subclass may also enable preferentialengagement of clearance or Ag modulation pathways. Similarly,although many different cells express FcgRs and therefore may beinvolved in Ag+ cellular clearance or Ag modulation (43), previ-ous studies suggest that various subsets of macrophages may beinvolved in RBC clearance (44), indicating that these cells maybe uniquely positioned to mediate Ag+ cell removal following Abengagement. However, whether these same cells engage both clear-ance and modulation pathways remains unknown, providing anotherpossible target that may facilitate differential induction of Ag mod-ulation versus clearance following Ab engagement in vivo. Futurestudies will consider these possibilities.In the current studies, using retransfer experiments, we dem-

onstrated that there is a subset of RBCs resistant to clearance byan FcgR-dependent process (see Fig. 2) and also showed that thekinetics of Ag modulation correlated to this clearance. Technicallimitations prevented us from unequivocally testing the interpre-tation that Ag modulation is responsible for resistance (i.e., to treatRBCs such that Ag modulation does not occur and determinewhether all of the RBCs then clear). However, our Ab titrationstudies indicate that the level of Ab bound after Ag modulationresults in decreased clearance (see Fig. 6). These data stronglysuggest that, indeed, Ag modulation is capable of rendering cellsresistant to clearance by FcgR-dependent pathways.The high level of Ag on Ag+ cells prior to transfer suggests that

all cells should be equally sensitive to clearance following en-gagement of Ab in vivo. However, only 40% of Ag+ cells rapidlyclear following transfer, with the remaining cells adopting a muchslower rate of clearance, consistent with the possible developmentof cellular resistance to Ab-mediated removal. As the FcgR sys-tem appears to be involved in both clearance and Ag modulation,the failure of recipients to clear 100% of cells following transferlikely reflects a dynamic process of simultaneous Ag modulation

FIGURE 9. Anti-Fy3 and anti-GPA induce specific modulation of their target Ags. (A) Clearance kinetics of double Ag+ cells (HOD+ and GPA+)

following transfer into nonimmunized, anti-GPA–immunized, or anti-Fy3–immunized recipients, as indicated. (B) Examination of bound Ab, detectable

Fy3 Ag, or detectable GPA Ag 2 d following transfer of double Ag+ cells (solid black line) or Ag2 cells (gray) into nonimmunized, anti-GPA–immunized,

or anti-Fy3–immunized recipients, as indicated. Representative flow plots are reflective of two independent experiments. Each experiment represents six

mice per group at each time point analyzed.


and clearance; cells that undergo Ag modulation escape the initialwave of clearance, whereas those that fail to rapidly undergo Agmodulation remain sensitive to clearance pathways and are re-moved from the circulation. However, the factors that dictatewhich cells will undergo Ag modulation versus clearance remainunknown and will be the subject of future studies.As many autoantibodies, alloantibodies, and Ab-based thera-

peutics engage FcgR (43, 45, 46), the present results not onlyprovide significant insight into potential regulatory pathways re-sponsible for modulating FcgR effector function but will likelyprovide a unique tool to facilitate additional insight into factorsthat dictate the consequence of FcgR engagement in vivo. Forexample, although reduced Ab effector function following Abengagement would likely modulate the outcome of cell survivalfavorably in settings of autoimmunity or alloimmunization, sim-ilar alterations would likely reduce the therapeutic efficacy of Ab-based therapeutics (25). As a result, a greater understanding of themechanisms and factors responsible for regulating Ab-induced Agloss have the potential to facilitate the development of novel ther-apeutics aimed at treating individuals with Ab-mediated diseasewhile also enabling the enhancement of Ab-based therapeutics.

AcknowledgmentsWe thank Krystal Hudson, Kate Henry, and Geetha Mylvaganamis for help-

ful discussions.

DisclosuresJ.C.Z. has sponsored research agreements with Immucor and Terumo, nei-

ther of which is related to the work in this study.

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