Antigen Modulation Confers Protection to
Red Blood Cells from Antibody through Fc γ
Receptor Ligation
This information is current as
of June 14, 2017.
Sean R. Stowell, Justine S. Liepkalns, Jeanne E.
Hendrickson, Kathryn R. Girard-Pierce, Nicole H. Smith, C.
Maridith Arthur and James C. Zimring
Supplementary
Material
References
Subscription
Permissions
Email Alerts
http://www.jimmunol.org/content/suppl/2013/10/09/jimmunol.130088
5.DC1
This article cites 45 articles, 18 of which you can access for free at:
http://www.jimmunol.org/content/191/10/5013.full#ref-list-1
Information about subscribing to The Journal of Immunology is online at:
http://jimmunol.org/subscription
Submit copyright permission requests at:
http://www.aai.org/About/Publications/JI/copyright.html
Receive free email-alerts when new articles cite this article. Sign up at:
http://jimmunol.org/alerts
The Journal of Immunology is published twice each month by
The American Association of Immunologists, Inc.,
1451 Rockville Pike, Suite 650, Rockville, MD 20852
Copyright © 2013 by The American Association of
Immunologists, Inc. All rights reserved.
Print ISSN: 0022-1767 Online ISSN: 1550-6606.
Downloaded from http://www.jimmunol.org/ by guest on June 14, 2017
J Immunol 2013; 191:5013-5025; Prepublished online 9
October 2013;
doi: 10.4049/jimmunol.1300885
http://www.jimmunol.org/content/191/10/5013
The Journal of Immunology
Antigen Modulation Confers Protection to Red Blood Cells
from 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*,‡
A
lthough adaptive immunity has the capacity to recognize
a vast range of antigenic determinants, this inherent plasticity also increases the risk of autoimmunity (1). As a
result, a distinct set of regulatory mechanisms evolved to aid
adaptive immunity in discriminating self from non-self (2, 3).
Although central and peripheral tolerance mechanisms reduce the
likelihood of self-reactivity, these regulatory programs occasionally fail to fully eliminate or inactivate self-reactive cells (4).
When tolerance mechanisms fail, the cellular targets of immunity
themselves can provide an additional layer of protection through
resistance to effector mechanisms. Such cellular protection often
reflects 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 selfeffector molecules that otherwise potently destroy many pathogens (8). Overall, these pathways are best understood in the
context of cell-mediated immunity [e.g., induction of anergy or
deletion in responding T cells and/or resistance to effector pathways 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.
*Department of Pathology and Laboratory Medicine, Emory University, Atlanta, GA
30322; †Department of Pediatrics, Aflac Cancer and Blood Disorders Center, Emory
University School of Medicine, Atlanta, GA 30032; and ‡Puget Sound Blood Center
Research Institute, Seattle, WA 98102
1
S.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 and
the Puget Sound Blood Center (to J.C.Z.).
Address correspondence and reprint requests to Dr. James C. Zimring, Puget Sound
Blood Center Research Institute, 1551 Eastlake Avenue E, Seattle, WA 98102. E-mail
address: [email protected]
The online version of this article contains supplemental material.
Abbreviations used in this article: DiD, 1,19-dioctadecyl-3,3,39,39-tetramethylindodicarbocyanine perchlorate; DiI, 1,19-dioctadecyl-3,3,3939-tetramethylindocarbocyanine
perchlorate; DiO, 3,39-dihexadecyloxacarbocyanine perchlorate; GPA, glycophorin A;
HOD, hen egg lysozyme–OVA–Duffy; KO, knockout; WT, wild-type.
Copyright Ó 2013 by The American Association of Immunologists, Inc. 0022-1767/13/$16.00
www.jimmunol.org/cgi/doi/10.4049/jimmunol.1300885
Although Abs in this case mediate the tissue damage, the Absecreting cells themselves often reside at distant sites from the
target 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 the
effects of Abs themselves. This strategy is not thematically different from CD8+ T cells resisting perforin- or granzyme-mediated
toxicity to escape issues of fratricide during cellular immune responses (11). However, as effector pathways for Abs are quite
distinct from those used by T cells, the particular mechanisms must
be suited to Ab-mediated damage.
Abs typically activate one or both of two primary effector mechanisms following engagement of Ag (14). Ab-induced complement activation results both in target opsonization (through C3
deposition) and in cellular lysis (through membrane attack complex
formation) (15). In addition, binding of Ab deposits Fcg domains,
which directly opsonize cellular targets through FcgRs (16). In the
case of complement, multiple resistance pathways have been described by which self-tissues prevent complement-mediated destruction, including both inhibition of complement activation and
facilitation of degradation of whatever complement may become
activated (8, 17, 18).
In contrast to the multiple and well-described mechanisms by
which self-tissues avoid damage from complement, it is less clear
if there are corresponding pathways to resist the effects of Fcg
domains ligating FcgRs. However, data in multiple systems suggest one pathway may exist. In particular, target tissues appear to
possess the ability to alter the autoantigen(s) or alloantigen(s)
being recognized such that Ab binding is substantially decreased
or eliminated. Such pathways have been observed for nicotinic
cholinergic receptors in myasthenia gravis (19, 20), Ags on platelets during idiopathic thrombocytopenic purpura (21), and Ags
on RBCs during autoimmune hemolytic anemia (22). Similar
pathways have been observed in response to alloantibodies as
well, which may play a role in transplanted organs “accommodating” over time to alloantibodies (23, 24). Finally, certain types
of neoplasia can co-opt the pathways to escape mAb therapy, such
as the selective shedding/shaving of CD20 by chronic lymphocytic
leukemia (25). For the purposes of the current report, this general
phenomenon will be termed “Ag modulation” and refers to an Ab-
Downloaded from http://www.jimmunol.org/ by guest on June 14, 2017
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.
5014
blood”) was mixed with blood collected from recipients of the first transfusion
at a 1:1 ratio with the surviving DiI-labeled HOD RBCs (“experienced HOD
blood”). The total mixture was brought to a 20% hematocrit with LPS-free
PBS, and 500 ml was transfused by tail-vein injection into fresh C57BL/6
mice.
RBC survival
After both the first and second transfusions, mice were anesthetized, and
25–50 ml blood was collected retro-orbitally at 2 h, 20 h, and 2 d posttransfusion. After the first transfusion, samples were analyzed by flow
cytometry, from which a ratio of DiI-labeled HOD blood to DiO-labeled
FVB blood within each animal was calculated. These ratios from Abtreated animals were then normalized to PBS-treated animals and graphed
as percent survival. After the second transfusion, the ratio of DiI-labeled
“experienced” HOD blood cells to DiO-labeled FVB blood cells was
calculated, and the resulting ratios were normalized to the ratios calculated
from 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 to
DiO-FVB RBC ratios were each normalized to their PBS counterparts
transfused with the same blood mixture (both originated from the same
first transfusion recipients). Survival graphs were all calculated and created
with Prism software. Error bars represent SD.
RBC characterization
A direct antiglobulin test was performed on samples collected after first and
second transfusions at a 1:100 dilution of goat anti-mouse Ig Abs (BD
Biosciences) or anti-C3 (Cedarlane). The samples were also stained with
a primary Ab, MIMA29, an anti-Fy6 Ab, or an anti-GPA Ab at a concentration 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 Pharmingen), diluted 1:100 in FACS buffer, as outlined previously (31). The
samples were also stained with Rat anti-TER119 (BD Biosciences Pharmingen), also diluted to 1:100 with FACS buffer; Rat IgG2b k (BD Biosciences Pharmingen) Abs were used as an isotype-matched control.
Materials and Methods
Results
Mice
Generation of an experimental model to Ag modulation as
a result of Ab engagement
C57BL/6 (B6), FVB, and C3 knockout (KO) mice were purchased from
The Jackson Laboratory (Bar Harbor, ME). FcgR KO mice (Fcer1g) were
purchased from Taconic Farms All mice were used at 8 to 14 wk of age.
HOD mice consisted of transgenic animals (FVB background) expressing
a 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 the
Emory University Department of Animal Resources Husbandry Services,
and all procedures were performed according to approved Institutional
Animal 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 Greg
Halverson at the New York Blood Center. Abs were purified by protein G
chromatography (Bio X Cell, West Lebanon, NH). The wild-type (WT)
C57BL/6 mice were passively immunized with 500 ml 200 mg MIMA29 in
PBS or 25 mg 6A7 in PBS, or were given PBS alone by tail-vein injection
2–5 h prior to transfusion.
Fluorescent labeling and transfusion of murine RBCs
HOD transgenic mice, HOD X GPA transgenic mice, and WT FVB mice
were anesthetized with isoflurane and exsanguinated by enucleation. Blood
collected was washed with PBS and labeled with one of three different
lipophilic dyes used to track the survival of these RBCs posttransfusion, as
outlined previously (29, 30). Briefly, HOD blood was typically labeled with
1,19-dioctadecyl-3,3,3939-tetramethylindocarbocyanine perchlorate (DiI), and
FVB blood was typically labeled with 3,39-dihexadecyloxacarbocyanine
perchlorate (DiO). After labeling, HOD and WT FVB blood cells were
mixed 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 the
above prepared blood mixture. For immunized mice receiving a second
transfusion, recipients first received unlabeled HOD blood followed by
a mixture of DiI-labeled HOD blood and DiO-labeled FVB blood 2 d later.
At 2 d posttransfusion, the mice were exsanguinated by enucleation, and
blood was mixed with freshly collected HOD blood labeled with a different
lipophilic dye, typically 1,19-dioctadecyl-3,3,39,39-tetramethylindodicarbocyanine perchlorate (DiD). The fresh DiD-labeled HOD blood (“fresh HOD
Our experimental system takes advantage of a transgenic mouse
that expresses a chimeric model Ag on RBCs, consisting of a fusion
of HEL to the C-terminal half of OVA to the human Duffy B
glycoprotein. The HOD Ag provides several well-defined immunological epitopes on the same protein, allowing a detailed study
of changes to an RBC surface protein during binding of different
Abs (see Fig. 1A for diagram of HOD Ag). To facilitate visualization and examination of HOD (Ag+) cells following adoptive transfer into HOD2 (Ag2) recipients, we labeled HOD (Ag+) cells with
the fluorescent dye DiI, to enable direct flow cytometric examination of Ag+ cells following Ab engagement in vivo (Fig. 1A). To
determine whether potential cellular alterations specifically occurred on Ag+ cells within a given recipient, a second population
of 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 detectable population for either DiO (Fig. 1B) or DiI (Fig. 1C).
Moreover, neither DiO nor DiI cells cross-fluoresced on the
channel of the other dye. Staining with anti-TER119, an RBCspecific Ag, confirmed that essentially 100% of events were
TER119+ after gating on DiI or DiO populations (Fig. 1B, 1C).
Finally, DiI-labeled Ag+ RBCs and DiO-labeled Ag2 RBCs were
mixed prior to adoptive transfer, and peripheral blood of recipients
was analyzed. Not only were the DiO+ and DiI+ populations easily
distinguishable, but the Ag+ HOD RBCs (DiI+) stained positive
with an Ab to HOD (anti-Fy3), whereas no staining was observed
on WT Ag2 RBCs (Fig. 1D). Thus, in aggregate, this technique
allows the introduction of RBCs into a recipient that has Abs
Downloaded from http://www.jimmunol.org/ by guest on June 14, 2017
induced alteration of recognized Ag as a mechanism to decrease
surface-bound Abs.
Ag modulation has been difficult to study in vivo, because the
target cells typically have ongoing synthesis of the target Ag, resulting in steady state equilibria that complicate analysis of the
fate of particular Ag molecules (21, 25). Moreover, as target cells
have ongoing division, tracking their numerical destruction over
time (or lack thereof) after Ab binding is likewise challenging (21,
25). To circumvent both of these limitations, we have made specific
use 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 population
into an Ag2 recipient allows examination of specific consequences,
including cellular clearance and the potential development of
protection against Ab-mediated removal, without the confounding variables of cell division or synthesis of additional protein. In
this study, we use a recently described RBC Ag–Ab system using
RBCs that express a unique model Ag: the hen egg lysozyme
(HEL)–OVA–Duffy (HOD) Ag (27, 28). Using this system, we
describe RBCs escaping destruction by Abs through Ag modulation. More importantly, the clearance mechanisms require FcgRs,
but not complement. This feature allows a mechanistic focus on
Ag modulation and its effects in an isolated FcgR-dependent pathway. We report that in addition to mediated clearance, FcgRs are
themselves instrumental in bringing about Ag modulation of Ab
targets. Taken together, these results elucidate details of a poorly
understood mechanism of cellular alteration in response to Ab
binding that may lead to protection against FcgR-mediated effector
responses in vivo.
Ag MODULATION PROTECTS RBCs FROM Abs
The Journal of Immunology
5015
against a defined Ag, combined with the ability to simultaneously
evaluate RBC survival and to visualize and characterize alterations
to the targeted Ag. Owing to the nature of RBCs, neither cellular
division nor new Ag synthesis is able to confound interpretation of
cellular survival or Ag modulation.
In vivo binding of Ab to RBC Ag and the effects on RBC
survival
To study the effects of Ab engagement on RBC survival, a mixure
of Ag2 (DiO) and Ag+ (DiI) RBCs was transferred i.v. into mice
that had been passively immunized with a mouse IgG2a mAb
against one epitope on the HOD Ag (anti-Fy3). In any given recipient, survival of Ag+ RBCs was normalized to survival of Ag2
RBCs, to allow an internal control for any variability in adoptive
transfer 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 antiFy3 to Ag+ RBCs, peripheral blood of recipients was stained with
anti-Ig and evaluated by flow cytometry. Anti-Ig was nonreactive
with either Ag2 or Ag+ RBCs in control mice injected with PBS
(Fig. 1E). In contrast, Ag+ RBCs in immunized mice were strongly
reactive with anti-Ig, whereas no signal was detected on Ag2 RBCs
(Fig. 1F). Together, these data demonstrate selective in vivo binding
of 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
Downloaded from http://www.jimmunol.org/ by guest on June 14, 2017
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 viability, 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.
5016
Ag2 RBCs in mice that received anti-Fy3 but had a normal survival in control animals that received PBS (Fig. 1G). However,
clearance kinetics were neither linear nor uniform. Although nearly
40% of Ag+ cells cleared within the first 2 h following adoptive
transfer, 2 d were required for an additional 40% to clear (Fig. 1G).
These clearance kinetics indicate an alteration to the biology regarding RBC clearance after 2 h.
RBCs surviving despite Ab binding are resistant to clearance
Rather, the majority of Ag+ RBCs cleared at the slower rate, consistent with the second phase of clearance (see Fig. 1). This result
was not due to simply transfusing a smaller number of RBCs as
a result of the rapid clearance phase eliminating 40% of RBCs, as
decreasing the RBC number of Ag+ RBCs during the first transfer
had the opposite effect (Supplemental Fig. 1). It was not possible
in 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 a
second mouse (Supplemental Fig. 1). However, we did control for
the possibility that slower clearance was due to just having circulated in a mouse (See Fig. 2A, group 2), which did not prevent the
rapid clearance phase in the second transfer but instead resulted in
enhanced clearance (Fig. 2D).
Although these results suggested that Ag+ cells may acquire
resistance to Ab-mediated removal, this approach did not specifically determine whether immunized recipients retain the capacity
to clear Ag+ cells following the development of reduced rates of
clearance. To test this, Ag+ cells were transferred into immunized
recipients, followed by a second transfer of Ag+ cells into the
same recipients 2 d later. As a control, immunized recipients not
previously exposed to Ag+ cells were transferred with Ag+ cells in
parallel (Fig. 3A). Ag+ cells experienced similar levels of clearance in immunized recipients that previously received Ag+ cells
as immunized recipients not previously exposed to Ag+ cells,
strongly suggesting that immunized recipients retain the ability to
remove Ag+ cells following the development of reduced rates of
clearance (Fig. 3B, 3C). As a whole, our interpretation of these
data is that Ab-bound HOD Ag+ RBCs that survive the rapid
clearance phase after initial transfer are resistant to subsequent
clearance.
Clearance of HOD RBCs by anti-Fy3 requires FcgRs, but
not C3
To further investigate mechanisms of clearance resistance, it was
necessary to establish the fundamental mechanisms of clearance
itself. 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.
Downloaded from http://www.jimmunol.org/ by guest on June 14, 2017
The existence of a rapid phase of RBC clearance followed by
significantly slower clearance raised the hypothesis that a subpopulation of RBCs may resist clearance mechanisms. However,
these findings were also compatible with alternative and more
trivial hypotheses, in particular, depletion of Ab or saturation of
RBC clearance mechanisms. To test these hypotheses, two separate
approaches were taken. In the first approach, a two-stage transfer
experiment was carried out (see Fig. 2A for diagram). In the first
transfer, a mixture of Ag+ (DiI) and Ag2 (DiO) RBCs was transferred into animals immunized with anti-Fy3 (as above), and
the rapid phase of RBC clearance was allowed to occur. At
2 d later, the RBCs that had survived were recovered by exsanguinating recipient mice. The recovered RBCs were then transferred to new mice (second transfer), which had been passively
immunized with anti-Fy3. In this way, surviving RBCs were exposed to a fresh dose of Ab and RBC clearance machinery that
had not yet consumed Ab-bound RBCs. The above hypotheses
predict that if the slowed clearance after 40% removal was due to
Ab depletion or saturation of clearance mechanisms, then a new
“rapid phase” should be observed during the second transfer. In
contrast, if the RBCs have a phenotype resistant to clearance, then
in the second transfer, they should have kinetics similar to those of
the 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 clearance phase (Fig. 2B). However, only a small and limited rapid
clearance phase was observed during the second transfer (Fig. 2C).
Ag MODULATION PROTECTS RBCs FROM Abs
The Journal of Immunology
5017
RBCs in vivo involve either FcgRs or complement. However,
alternative pathways have also been described that do not require
either FcgRs or complement (28). Thus, to evaluate clearance
mechanisms of anti-Fy3 and HOD, transfer experiments were
carried out in mice with targeted deletions of either the C3 component of complement or the common g-chain of FcgRs.
C3 is the main complement opsonin that causes RBC consumption and is also instrumental to downstream formation of the
membrane attack complex. Clearance in C3 KO mice had a kinetics
and magnitude similar to that observed in WT recipients (Fig. 4A).
To test whether C3 is deposited on Ag+ RBCs after anti-Fy3
binding, peripheral blood samples were obtained at 2 h posttransfusion from WT recipients and were stained with anti-C3.
Anti-C3 reactivity was detected in the presence of anti-Fy3 in
a population of Ag+ (DiI) RBCs (Fig. 4B). This C3 deposition
required the presence of the target Ag, as no C3 was detected on
Ag2 RBCs (DiO) (Fig. 4B). The deposition of C3 on HOD RBCs
was not a spontaneous event or a function of the nonclassical
pathway, as no C3 was detected on either Ag+ or Ag2 RBCs in
control mice that received PBS (Fig. 4B). Thus, deposition of C3
in this system requires that Ag and Ab be simultaneously present.
Representative flow plots during clearance in the C3 KO mice are
shown and, as predicted owing to the targeted deletion of C3, no
anti-C3 reactivity is observed in either group (Fig. 4C). Together,
these data indicate that C3 is indeed deposited on the surface of
some HOD RBCs after binding of anti-Fy3; however, the functional effect of C3 binding is unclear with regard to clearance, as
clearance curves are normal in the C3 KO animal. Thus, these data
do not rule out the involvement of C3 but do demonstrate that C3
is not required for clearance in this system.
Although C3 engagement is thought to be the primary complement effector pathway whereby Abs initiate complement, several C3-independent pathways of complement activation have
been described (32), suggesting that downstream targets of com-
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 antiFy3 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.
Downloaded from http://www.jimmunol.org/ by guest on June 14, 2017
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.
5018
RBCs resistant to clearance undergo Ag modulation of surface
HOD that correlates with resistance to clearance
To further characterize Ag–Ab interactions on the RBCs, we directly examined Ag+ cells for Ab engagement at various points
following adoptive transfer into immunized recipients. Peripheral
blood was collected at the indicated time points, stained with antiIg, and analyzed by flow cytometry (Fig. 5A). Although the level
of bound Ab remained high during the initial rapid phase of
clearance, the amount of detectable Ab on Ag+ cells progressively
decreased over time (Fig. 5A). The staining was specific for the
presence of anti-Fy3, as no signal was detected on Ag2 RBCs
(Fig. 5A) or on either Ag+ or Ag2 RBCs in control mice that
received PBS (Fig. 5A and Supplemental Fig. 3).
As immunization of recipients reflected passive transfer of Ab,
it remained possible that reduced Ab engagement simply reflected
rapid consumption of passively administered Ab. However, staining
of recovered RBCs with anti-Fy3 itself, followed by anti-Ig, failed
to increase the signal, indicating that the available anti-Fy3 binding
sites were saturated (Fig. 5B). In contrast to the observed decrease
in Ag seen on Ag+ RBCs in anti-Fy3–immunized mice, Ag+ RBCs
recovered after transfer into PBS-treated mice retained full staining
(Fig. 5B, Supplemental Fig. 3). Apparent loss of detectable Ag did
not appear to reflect wholesale loss of the plasma membrane, as
cells experiencing Ag modulation isolated from immunized recipients displayed forward scatter profiles very similar to those of
Ag+ cells transferred into nonimmunized controls (Fig. 5C, 5D).
Together, these data indicate that the amount of detectable Ag on
the transferred Ag+ RBCs decreases as a function of being transferred into an animal with circulating anti-Fy3, and establishes the
phenomenon 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 engagement on Ag+ cells paralleled the decreased kinetics of RBC
clearance and the generation of RBC resistance to clearance (see
Figs. 1G, 2). These observations led us to hypothesize that alterations in surface RBC Ag, and thus decreased Ab engagement,
was involved with decreased RBC clearance. However, it is important to note that although Ag modulation decreases detectable
Ag, Ag-bound Ab remains readily detectable. Thus, for Ag modulation to have an effect on RBC clearance, there would have
to be a density dependence of FcgR ligation. To test this hy-
pothesis, we incubated cells with varying levels of Ab or no Ab
in vitro prior to adoptive transfer in an effort to recapitulate the
potential impact of reduced Ab levels on Ag+ cell removal (Fig.
6A). Incubation of Ag+ cells with Ab in vitro enabled isolation of
RBCs with Ag saturation [hi], an Ab intermediate population [int],
or cells with no Ab [0] prior to transfer (Fig. 6B, Supplemental
Fig. 3). Each population of RBCs was then transferred into WT
mice (no passive Ab into the mice), and mice were bled at the
indicated time. The amount of surface IgG was determined by
staining with anti-Ig, whereas the amount of Fy3 Ag was determined by staining with anti-Fy3 followed by anti-Ig.
Compared with control HOD RBCs that were transferred without preincubation with Ab (Fig. 6C, top row), transfer of Absaturated cells [hi] displayed significant decrease in bound Ab
over time (Fig. 6C, bottom row). Likewise, staining with anti-Fy3
followed by anti-Ig demonstrated decreased detectable Ag in [hi]
RBCs (Fig. 6D, bottom row). This observed pattern in [hi] RBCs
was similar to what was observed following adoptive transfer of
Ag+ cells into immunized recipients (see Fig. 5). In contrast, [int]
cells failed to display a similar decrease in surface Ab from pretransfer levels (Fig. 6C, middle row). Moreover, staining with antiFy3 and anti-Ig increased the signal on [int] cells to a level similar
to that in control HOD RBCs that were transferred without preincubation with Ab (Fig. 6D, middle and top rows; Supplemental
Fig. 3). Thus high levels of Ab, but not intermediate levels, result
in 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 enumerated
by the same DiI/DiO techniques used above. Whereas [hi] levels of
Ab caused rapid clearance, [int] levels of Ab caused substantially
slower clearance (Fig. 6E). The [hi] and [int] Ab incubations were
titrated to mimic Ab levels on Ag+ RBCs prior to transfer and after
the rapid clearance phase, respectively. Thus, these findings are
consistent with Ag modulation, resulting in a sufficient decrease in
surface Ab to render RBCs resistant to clearance. Together, these
data indicate that both clearance and Ag modulation are a function
of 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 clearance
of transferred HOD RBCs, this does not exclude its possible role
in Ag modulation. Indeed, Taylor et al. (34) have reported that C3
deposition on Ig itself can prevent binding of secondary Abs through
direct blocking or steric hindrance. To test this hypothesis, Ag
modulation was evaluated after transfer into C3 KO mice. Transfer
of Ag+ cells into immunized C3 KO mice resulted in a decrease in
detectable Ag similar to that in WT recipients (Fig. 7A, 7B).
Similar results were obtained following transfer of Ag+ cells into
C5-deficient FVB immunized recipients (Supplemental Fig. 4).
These data indicate that C3 and C5 are not required for Ag modulation in this system, and we thus reject the hypothesis that the
mechanism 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 immunized
recipients. In the absence of FcgRs, Ab persisted on cells unaltered over extended periods (Fig. 7C). As above, RBCs did not
clear in FcgR mice that were immunized with anti-Fy3, whereas
normal clearance occurred in WT animals (Fig. 7D–F). These data
indicate that FcgRs are required for the process of Ag modulation.
Decrease in detectable Ag extends intra- but not
intermolecularly
To determine whether Ag modulation was specific to the epitope
recognized by anti-Fy3, we compared levels of Fy3 with an epitope
Downloaded from http://www.jimmunol.org/ by guest on June 14, 2017
plement activation may be involved in cellular clearance. To test
this, we next transferred Ag+ cells into immunized FVB recipients, which are deficient in the C5 component of complement
required for initiation of the membrane attack complex (33). Immunized FVB recipients displayed a similar ability to clear Ag+
cells (Supplemental Fig. 2), strongly suggesting that Ag+ cell
clearance occurs independent of the downstream complement
effector molecules. Thus, these data do not rule out the involvement of C3 or C5 but do demonstrate that C3 and C5 are not
individually required for clearance in this system.
To determine the role of FcgRs in this process, the same experiment was carried out in mice with a targeted deletion of the
common Fc g-chain, resulting in a lack of functional FcgRI,
FcgRIII, and FcgRIV (16). No clearance of Ag+ cells was observed following transfer into immunized FcgR KO recipients
(Fig. 4D), indicating that FcgRs are required for RBC clearance in
this system. Because essentially no clearance was observed in the
FcgR KO mice, this also suggests that complement is not sufficient to induce RBC clearance. Together, these data indicate that
FcgRs are required for clearance of HOD RBCs by anti-Fy3 and
that C3 is neither required nor sufficient for clearance.
Ag MODULATION PROTECTS RBCs FROM Abs
The Journal of Immunology
5019
Downloaded from http://www.jimmunol.org/ by guest on June 14, 2017
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.
on the opposite end of HOD (Fy6; see Fig. 8A). Ag+ cells were
isolated at various time points following transfer and were incubated with an anti-Fy6 Ab, followed by detection of total Ab
bound to cells, similar to the method used to determine whether
alterations in the Fy3 target epitope occurred over time. Although
the Fy6 epitope remained detectable, with unaltered levels of Ag
on cells transferred into nonimmunized recipients, the Fy6 epitope
experienced a decrease similar to that observed for the Fy3 epitope over time in immunized mice (Fig. 8B–D). These data indicate that a separate epitope on the same molecule, and not recognized
by anti-Fy3, undergoes a simultaneous and similar decrease in detectability.
To test whether Ag modulation was generalized to epitopes on
separate molecules, two approaches were taken. In the first approach, we tested the expression of Ter119, an RBC-specific Ag
unrelated to HOD. Ter119 RBC Ag expression remained unchanged
over the same time and under the same conditions (Fig. 8E). In the
second approach, we crossed HOD transgenics by transgenics
expressing the human glycophorin A (GPA) Ag, a human Ag similar
to the mouse Ter119 Ag, on RBCs and transferred these double
5020
Ag MODULATION PROTECTS RBCs FROM Abs
Downloaded from http://www.jimmunol.org/ by guest on June 14, 2017
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.
positive cells into nonimmunized, anti-Fy3–immunized, or antiGPA–immunized recipients. Although nonimmunized recipients
failed to clear double Ag+ cells, anti-Fy3–immunized and antiGPA–immunized recipients each effectively cleared double positive
cells (Fig. 9A), consistent with previous results and those of this
study (35). Examination of bound Ab and detectable Ag demonstrated that Ab could be detected only on double Ag+ cells following
transfer into immunized recipients and that double positive RBCs
experienced modulation of HOD Ag, but not GPA Ag, following
transfer into anti-Fy3–immunized recipients (Fig. 9B). Conversely,
transfer of double Ag+ cells into anti-GPA–immunized recipients
likewise resulted in Ag modulation of the GPA Ag, whereas the
HOD Ag remained unaltered (Fig. 9B). Together, these data indicate
that a decrease in detectable Ag appears to reflect specific modulation of the target Ag following Ab engagement.
Discussion
Innate immunity and adaptive immunity have evolved multiple
effector pathways to protect the host from pathological microbes.
Abs represent one such pathway and have the capacity to destroy
the targets they bind through both fixation of complement and
ligation of FcgRs. However, the presence of such Abs necessitates
an intrinsic risk of damaging self-tissues. As an evolutionary response to this damage, multiple pathways have been described
The Journal of Immunology
5021
by which self-tissues can inactivate complement, thus mitigating
damage from autoantibodies or alloantibodies. In contrast, little
has been described regarding FcgR-mediated pathways and the
extent to which protection pathways have evolved. One pathway
that has been put forward is referred to in this article as “Ag
modulation,” a process by which target Ags change so as to decrease Ab binding and thus limit FcgR ligation. Detailed analysis
of the effects of Ag modulation requires a system in which FcgRs
are isolated as an Ab effector pathway (i.e., complement does not
play a role). Moreover, one must control issues of cellular proliferation and Ag resynthesis by target cells during any ongoing
Ag modulation. In this article, we describe just such a system by
taking advantage of RBCs, which neither proliferate nor synthesize protein and are cleared by an FcgR dependent/complementindependent pathway.
The data presented generate novel understanding of Ag modulation at several different points. First, the presence of both
anti-Fy3 and FcgRs is required for both RBC clearance and Ag
modulation. Hence, the biology and the multiple effects of Abinduced FcgR ligation overlap. One trivial explanation that might
come from these observations would be that RBCs with a baseline
level of higher Ag expression are cleared, whereas those with a
baseline lower Ag expression are not. Thus, what appears to be
ongoing Ag modulation on RBCs actually just represents selective
removal of a population with high Ag expression. However, we
reject this interpretation as mathematically inconsistent with
staining of RBCs at baseline. After 2 h, 50% of the Ag+ RBCs are
still circulating and have a 5-fold decrease in surface Ag. However, prior to transfer into immunized animals, only a very small
percentage of RBCs have lower baseline levels of Ag on the
surface (see Fig. 1). Accordingly, our interpretation is that surviving RBCs started with high levels of detectable Ag and underwent an Ag modulation process that substantially decreased
detectable Ag.
In the above context, the simultaneous requirement for FcgR
to carry out both clearance and Ag modulation is a unique and
Downloaded from http://www.jimmunol.org/ by guest on June 14, 2017
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.
5022
Ag MODULATION PROTECTS RBCs FROM Abs
novel finding. This indicates an FcgR-dependent biology that
results in the modulation of presynthesized Ag on the RBC surface. The precise details of the mechanism by which this occurs
are not clear at all levels; however, we can make some distinct
conclusions based upon the current data. First, according to published literature, C3 that deposits on cell-bound Ab can modify it
such that anti-Ig no longer recognizes the Ab (34, 36, 37). Given
that C3 deposition was detected after anti-Fy3 bound to Ag+ RBCs
(see Fig. 3), and given the known phenomenon, this seemed
a likely hypothesis. However, we reject this hypothesis based upon
the fact that Ag modulation occurs unabated in C3 KO mice.
Similar results were observed following transfer into immunized
FVB C5-deficient recipients (33). This observation does not address the more general issue of any modification of anti-Fy3 (other
than involving C3 or C5) that may render it undetectable by antiIg. However, the observation that both Fy6 and Fy3 undergo Ag
modulation after exposure to anti-Fy3 provides additional evidence that simple modification of the anti-Fy3 is not a likely
mechanism. Together, these data indicate that the mechanism of
Ag modulation is not modification of the Ag-bound Ab, and likely
represents 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 the
cellular membrane, partial removal of select epitopes, or protein
modification so as to render the Ag undetectable by Ab. The
modulation of Fy6 by anti-Fy3 rejects the hypothesis that anti-Fy3
is causing an epitope-selective destruction of the protein. Moreover,
reduction of the protein and chemical denaturation seem unlikely
mechanisms, as both anti-Fy3 and anti-Fy6 recognize linear peptide
sequences. In addition, the selective modulation of the Fy3 Ag following transfusion of HOD X GPA RBCs also suggests that anti-Fy3
engagement results in specific alterations to the target Ag. Although
complete removal of a transmembrane protein from the RBC surface,
without damaging the RBC, may seem unlikely, at least one human
case of Ag modulation during autoimmune hemolytic anemia provided substantial evidence that the Ag was removed from the RBC
(38). This removal would not necessarily require pulling the protein 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 CD20
by chronic lymphocytic leukemia in response to mAb immunotherapy (i.e., rituximab) (25). As some cases of autoimmune hemolytic anemia, in which changes in cell size may be detectable,
reflect Ab engagement of high-density Ags (39), the lack of detectable alterations in cell size may simply reflect lower Ag density
in the present system with a corresponding reduction in the amount
of membrane removed. As a result, subtle changes in Ag levels, as
observed following Ag modulation, may not produce enough loss
of membrane to be detected with the tools used in this study. As a
result, we propose a model by which Ab binding Ag and ligating
FcgR 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 occurs
by several different mechanisms, depending upon the nature of the
Ab, the Ag, and the target cell. Very much as in humans, the same
diversity of mechanisms appears to be present in mice. We have
previously reported that Ag modulation of murine HEL on RBCs
requires the simultaneous binding of two separate Abs that bind to
distinct epitopes. However, Abs that engage HEL in this system fail
to induce RBC clearance, making it difficult to determine whether
Ag modulation can protect cells from Abs capable of inducing RBC
removal. However, such appears not to be the case in the current
Downloaded from http://www.jimmunol.org/ by guest on June 14, 2017
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.
The Journal of Immunology
5023
setting, as a monoclonal anti-Fy3 resulted in Ag modulation and
clearance. Similarly, the ability of anti-GPA to induce Ag modulation demonstrates that Ag modulation is not limited to anti-Fy3.
The unique biphasic clearance pattern induced by anti-GPA is
consistent with previous studies (35). Given the potential involvement of complement in anti-GPA–mediated clearance (40),
complement receptors may initially trap RBCs following Abmediated complement deposition and then release some RBCs
following subsequent complement degradation, as suggested by
previous studies examining the consequences of complement deposition on RBCs (36, 41). Although additional studies are needed
to further understand the mechanisms of anti-GPA–induced clearance and Ag modulation, these results suggest that alterations in
Ag following Ab engagement may reflect a general mechanism
of cellular protection from Ab-mediated removal. Thus, although
the current findings cannot be used to draw generalized conclusions regarding Ag modulation, Ag modulation appears to reflect a general phenomenological descriptor that captures several
different processes with a common outcome. However, to the best
of our knowledge, the current report represents the first description of a new pathway of Ag modulation, in which FcgRs are required 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 Ag
modulation remains unknown. Previous studies suggest that IgG2a
Abs preferentially engage FcgRIV, whereas IgG1 and IgG3 prefer
to bind FcgRIII and FcgRI, respectively (42, 43). As FcgR KO
recipients were used in the current study, it remains possible that
the IgG2a anti-Fy3 used in this study preferentially triggers
FcgRIVs following engagement of Ag+ cells in vivo. In contrast,
class switch variants of anti-Fy3 may preferentially engage alternative FcgRs. As it remains possible that the FcgRs responsible
for clearance may be different from those that induce Ag modu-
lation, manipulation of Ab subclass may also enable preferential
engagement of clearance or Ag modulation pathways. Similarly,
although many different cells express FcgRs and therefore may be
involved in Ag+ cellular clearance or Ag modulation (43), previous studies suggest that various subsets of macrophages may be
involved in RBC clearance (44), indicating that these cells may
be uniquely positioned to mediate Ag+ cell removal following Ab
engagement. However, whether these same cells engage both clearance and modulation pathways remains unknown, providing another
possible target that may facilitate differential induction of Ag modulation versus clearance following Ab engagement in vivo. Future
studies will consider these possibilities.
In the current studies, using retransfer experiments, we demonstrated that there is a subset of RBCs resistant to clearance by
an FcgR-dependent process (see Fig. 2) and also showed that the
kinetics of Ag modulation correlated to this clearance. Technical
limitations prevented us from unequivocally testing the interpretation that Ag modulation is responsible for resistance (i.e., to treat
RBCs such that Ag modulation does not occur and determine
whether all of the RBCs then clear). However, our Ab titration
studies indicate that the level of Ab bound after Ag modulation
results in decreased clearance (see Fig. 6). These data strongly
suggest that, indeed, Ag modulation is capable of rendering cells
resistant 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 engagement of Ab in vivo. However, only 40% of Ag+ cells rapidly
clear following transfer, with the remaining cells adopting a much
slower rate of clearance, consistent with the possible development
of cellular resistance to Ab-mediated removal. As the FcgR system appears to be involved in both clearance and Ag modulation,
the failure of recipients to clear 100% of cells following transfer
likely reflects a dynamic process of simultaneous Ag modulation
Downloaded from http://www.jimmunol.org/ by guest on June 14, 2017
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.
5024
Acknowledgments
We thank Krystal Hudson, Kate Henry, and Geetha Mylvaganamis for helpful discussions.
Disclosures
J.C.Z. has sponsored research agreements with Immucor and Terumo, neither of which is related to the work in this study.
References
1. Goodnow, C. C., J. Sprent, B. Fazekas de St Groth, and C. G. Vinuesa. 2005.
Cellular and genetic mechanisms of self tolerance and autoimmunity. Nature
435: 590–597.
2. Hogquist, K. A., T. A. Baldwin, and S. C. Jameson. 2005. Central tolerance:
learning self-control in the thymus. Nat. Rev. Immunol. 5: 772–782.
3. Hardy, R. R., and K. Hayakawa. 2001. B cell development pathways. Annu. Rev.
Immunol. 19: 595–621.
4. Walker, L. S., and A. K. Abbas. 2002. The enemy within: keeping self-reactive
T cells at bay in the periphery. Nat. Rev. Immunol. 2: 11–19.
5. Toscano, M. A., G. A. Bianco, J. M. Ilarregui, D. O. Croci, J. Correale,
J. D. Hernandez, N. W. Zwirner, F. Poirier, E. M. Riley, L. G. Baum, and
G. A. Rabinovich. 2007. Differential glycosylation of TH1, TH2 and TH-17
effector cells selectively regulates susceptibility to cell death. Nat. Immunol.
8: 825–834.
6. Cerliani, J. P., S. R. Stowell, I. D. Mascanfroni, C. M. Arthur, R. D. Cummings,
and G. A. Rabinovich. 2011. Expanding the universe of cytokines and pattern
recognition receptors: galectins and glycans in innate immunity. J. Clin.
Immunol. 31: 10–21.
7. Stowell, S. R., S. Karmakar, C. M. Arthur, T. Ju, L. C. Rodrigues, T. B. Riul,
M. Dias-Baruffi, J. Miner, R. P. McEver, and R. D. Cummings. 2009. Galectin-1
induces reversible phosphatidylserine exposure at the plasma membrane. Mol.
Biol. Cell 20: 1408–1418.
8. Zipfel, P. F., and C. Skerka. 2009. Complement regulators and inhibitory proteins. Nat. Rev. Immunol. 9: 729–740.
9. Steinman, R. M., D. Hawiger, and M. C. Nussenzweig. 2003. Tolerogenic
dendritic cells. Annu. Rev. Immunol. 21: 685–711.
10. Hiruma, K., H. Nakamura, P. A. Henkart, and R. E. Gress. 1992. Clonal deletion
of postthymic T cells: veto cells kill precursor cytotoxic T lymphocytes. J. Exp.
Med. 175: 863–868.
11. Müller, C., and J. Tschopp. 1994. Resistance of CTL to perforin-mediated lysis.
Evidence for a lymphocyte membrane protein interacting with perforin. J.
Immunol. 153: 2470–2478.
12. Lehmann, C., M. Zeis, N. Schmitz, and L. Uharek. 2000. Impaired binding of
perforin on the surface of tumor cells is a cause of target cell resistance against
cytotoxic effector cells. Blood 96: 594–600.
13. Cyster, J. G., K. M. Ansel, V. N. Ngo, D. C. Hargreaves, and T. T. Lu. 2002.
Traffic patterns of B cells and plasma cells. Adv. Exp. Med. Biol. 512: 35–41.
14. Ravetch, J. V., and R. A. Clynes. 1998. Divergent roles for Fc receptors and
complement in vivo. Annu. Rev. Immunol. 16: 421–432.
15. Walport, M. J. 2001. Complement. First of two parts. N. Engl. J. Med. 344:
1058–1066.
16. Nimmerjahn, F., and J. V. Ravetch. 2006. Fcgamma receptors: old friends and
new family members. Immunity 24: 19–28.
17. Ferreira, V. P., and M. K. Pangburn. 2007. Factor H mediated cell surface protection from complement is critical for the survival of PNH erythrocytes. Blood
110: 2190–2192.
18. Moller, G. 1964. Isoantibody-induced cellular resistance to immune haemolysis
in vivo and in vitro. Nature 202: 357–359.
19. Kao, I., and D. B. Drachman. 1977. Myasthenic immunoglobulin accelerates
acetylcholine receptor degradation. Science 196: 527–529.
20. Tzartos, S. J., D. Sophianos, and A. Efthimiadis. 1985. Role of the main immunogenic region of acetylcholine receptor in myasthenia gravis. An Fab
monoclonal antibody protects against antigenic modulation by human sera.
J. Immunol. 134: 2343–2349.
21. Boylan, B., H. Chen, V. Rathore, C. Paddock, M. Salacz, K. D. Friedman,
B. R. Curtis, M. Stapleton, D. K. Newman, M. L. Kahn, and P. J. Newman. 2004.
Anti-GPVI-associated ITP: an acquired platelet disorder caused by autoantibodymediated clearance of the GPVI/FcRgamma-chain complex from the human
platelet surface. Blood 104: 1350–1355.
22. Vengelen-Tyler, V., B. Gonzalez, G. Garratty, C. Kruppe, C. L. Johnson,
K. A. Mueller, and W. L. Marsh. 1987. Acquired loss of red cell Kell antigens.
Br. J. Haematol. 65: 231–234.
23. Bach, F. H., C. Ferran, P. Hechenleitner, W. Mark, N. Koyamada, T. Miyatake,
H. Winkler, A. Badrichani, D. Candinas, and W. W. Hancock. 1997. Accommodation of vascularized xenografts: expression of “protective genes” by donor
endothelial cells in a host Th2 cytokine environment. Nat. Med. 3: 196–204.
24. Park, W. D., J. P. Grande, D. Ninova, K. A. Nath, J. L. Platt, J. M. Gloor, and
M. D. Stegall. 2003. Accommodation in ABO-incompatible kidney allografts,
a novel mechanism of self-protection against antibody-mediated injury. Am. J.
Transplant. 3: 952–960.
25. Beum, P. V., A. D. Kennedy, M. E. Williams, M. A. Lindorfer, and R. P. Taylor.
2006. The shaving reaction: rituximab/CD20 complexes are removed from
mantle cell lymphoma and chronic lymphocytic leukemia cells by THP-1
monocytes. J. Immunol. 176: 2600–2609.
26. Mohandas, N., and P. G. Gallagher. 2008. Red cell membrane: past, present, and
future. Blood 112: 3939–3948.
27. Desmarets, M., C. M. Cadwell, K. R. Peterson, R. Neades, and J. C. Zimring.
2009. Minor histocompatibility antigens on transfused leukoreduced units of red
blood cells induce bone marrow transplant rejection in a mouse model. Blood
114: 2315–2322.
28. Liepkalns, J. S., E. A. Hod, S. R. Stowell, C. M. Cadwell, S. L. Spitalnik, and
J. C. Zimring. 2012. Biphasic clearance of incompatible red blood cells through
a novel mechanism requiring neither complement nor Fcgamma receptors in
a murine model. Transfusion 52: 2631–2645.
29. Stowell, S. R., K. L. Henry, N. H. Smith, K. E. Hudson, G. R. Halverson,
J. C. Park, A. M. Bennett, K. R. Girard-Pierce, C. M. Arthur, S. T. Bunting,
J. C. Zimring, and J. E. Hendrickson. 2013. Alloantibodies to a paternally derived RBC KEL antigen lead to hemolytic disease of the fetus/newborn in
a murine model. Blood 122: 1494–1504,
30. Liepkalns, J. S., C. M. Cadwell, S. R. Stowell, E. A. Hod, S. L. Spitalnik, and
J. C. Zimring. 2013. Resistance of a subset of red blood cells to clearance by
antibodies in a mouse model of incompatible transfusion. Transfusion 53: 1319–
1327.
31. Stowell, S. R., N. H. Smith, J. C. Zimring, X. Fu, A. F. Palmer, J. Fontes, U.
Banerjee, and M. H. Yazer. 2013. Addition of ascorbic acid solution to stored
murine red blood cells increases posttransfusion recovery and decreases
microparticles and alloimmunization. Transfusion 2013 Feb 5. doi: 10.1111/
trf.12106.
32. Krisinger, M. J., V. Goebeler, Z. Lu, S. C. Meixner, T. Myles, E. L. Pryzdial, and
E. M. Conway. 2012. Thrombin generates previously unidentified C5 products
that support the terminal complement activation pathway. Blood 120: 1717–
1725.
33. Wong, K., S. Bumpstead, L. Van Der Weyden, L. G. Reinholdt, L. G. Wilming,
D. J. Adams, and T. M. Keane. 2012. Sequencing and characterization of the
FVB/NJ mouse genome. Genome Biol. 13: R72.
34. Beum, P. V., A. D. Kennedy, Y. Li, A. W. Pawluczkowycz, M. E. Williams, and
R. P. Taylor. 2004. Complement activation and C3b deposition on rituximabopsonized cells substantially blocks binding of phycoerythrin-labeled antimouse IgG probes to rituximab. J. Immunol. Methods 294: 37–42.
35. Hod, E. A., C. M. Cadwell, J. S. Liepkalns, J. C. Zimring, S. A. Sokol,
D. A. Schirmer, J. Jhang, and S. L. Spitalnik. 2008. Cytokine storm in a mouse
model of IgG-mediated hemolytic transfusion reactions. Blood 112: 891–894.
36. Stowell, S. R., A. M. Winkler, C. L. Maier, C. M. Arthur, N. H. Smith,
K. R. Girard-Pierce, R. D. Cummings, J. C. Zimring, and J. E. Hendrickson.
2012. Initiation and regulation of complement during hemolytic transfusion
reactions. Clin. Dev. Immunol. 2012: 307093.
37. Girard-Pierce, K. R., S. R. Stowell, N. H. Smith, C. M. Arthur, H. C. Sullivan,
J. E. Hendrickson, and J. C. Zimring. 2013. A novel role for C3 in antibodyinduced red blood cell clearance and antigen modulation. Blood. 122: 1793–
1801.
38. Williamson, L. M., J. Poole, C. Redman, N. Clark, Y. W. Liew, D. C. Russo,
S. Lee, M. E. Reid, and A. J. Black. 1994. Transient loss of proteins carrying
Kell and Lutheran red cell antigens during consecutive relapses of autoimmune
thrombocytopenia. Br. J. Haematol. 87: 805–812.
39. Victoria, E. J., S. W. Pierce, M. J. Branks, and S. P. Masouredis. 1990. IgG red
blood cell autoantibodies in autoimmune hemolytic anemia bind to epitopes on
red blood cell membrane band 3 glycoprotein. J. Lab. Clin. Med. 115: 74–88.
40. Schirmer, D. A., S. C. Song, J. P. Baliff, S. O. Harbers, R. A. Clynes, A. KropWatorek, G. R. Halverson, M. Czerwinski, and S. L. Spitalnik. 2007. Mouse
models of IgG- and IgM-mediated hemolysis. Blood 109: 3099–3107.
Downloaded from http://www.jimmunol.org/ by guest on June 14, 2017
and clearance; cells that undergo Ag modulation escape the initial
wave of clearance, whereas those that fail to rapidly undergo Ag
modulation remain sensitive to clearance pathways and are removed from the circulation. However, the factors that dictate
which cells will undergo Ag modulation versus clearance remain
unknown and will be the subject of future studies.
As many autoantibodies, alloantibodies, and Ab-based therapeutics engage FcgR (43, 45, 46), the present results not only
provide significant insight into potential regulatory pathways responsible for modulating FcgR effector function but will likely
provide a unique tool to facilitate additional insight into factors
that dictate the consequence of FcgR engagement in vivo. For
example, although reduced Ab effector function following Ab
engagement would likely modulate the outcome of cell survival
favorably in settings of autoimmunity or alloimmunization, similar alterations would likely reduce the therapeutic efficacy of Abbased therapeutics (25). As a result, a greater understanding of the
mechanisms and factors responsible for regulating Ab-induced Ag
loss have the potential to facilitate the development of novel therapeutics aimed at treating individuals with Ab-mediated disease
while also enabling the enhancement of Ab-based therapeutics.
Ag MODULATION PROTECTS RBCs FROM Abs
The Journal of Immunology
41. Brown, D. L., P. J. Lachmann, and J. V. Dacie. 1970. The in vivo behaviour of
complement-coated red cells: studies in C6-deficient, C3-depleted and normal
rabbits. Clin. Exp. Immunol. 7: 401–421.
42. Nimmerjahn, F., A. Lux, H. Albert, M. Woigk, C. Lehmann, D. Dudziak,
P. Smith, and J. V. Ravetch. 2010. FcgRIV deletion reveals its central role for
IgG2a and IgG2b activity in vivo. Proc. Natl. Acad. Sci. USA 107: 19396–19401.
43. Hogarth, P. M. 2002. Fc receptors are major mediators of antibody based inflammation in autoimmunity. Curr. Opin. Immunol. 14: 798–802.
5025
44. Hendrickson, J. E., T. E. Chadwick, J. D. Roback, C. D. Hillyer, and
J. C. Zimring. 2007. Inflammation enhances consumption and presentation of
transfused RBC antigens by dendritic cells. Blood 110: 2736–2743.
45. Sibéril, S., C. A. Dutertre, W. H. Fridman, and J. L. Teillaud. 2007. FcgammaR:
The key to optimize therapeutic antibodies? Crit. Rev. Oncol. Hematol. 62: 26–
33.
46. Turgeon, N. A., A. D. Kirk, and N. N. Iwakoshi. 2009. Differential effects of
donor-specific alloantibody. Transplant. Rev. (Orlando) 23: 25–33.
Downloaded from http://www.jimmunol.org/ by guest on June 14, 2017
SUPPLEMENTAL FIGURE LEGENDS:
Supplemental Figure 1 RBC clearance and antibody engagement after transfer Ag+
cells into immunized recipients. (A) —ƒ–‹ˆ‹…ƒ–‹‘ ‘ˆ ‰Ϊ …‡ŽŽ …Ž‡ƒ”ƒ…‡ ʹ Š‘—”•
ˆ‘ŽŽ‘™‹‰ –”ƒ•ˆ‡” ‘ˆ ͷͲͲ μŽ …‘–ƒ‹‹‰ ʹͲΨ …– ȋȌ ‘” ͷͲͲ μŽ …‘–ƒ‹‹‰ ͲǤʹΨ
…–ȋŽ‘Ȍ‹–‘‘Ǧ‹—‹œ‡†‘”‹—‹œ‡†”‡…‹’‹‡–•(B)Ž‘™…›–‘‡–”‹…
‡šƒ‹ƒ–‹‘‘ˆ‰Ϊ…‡ŽŽ•ȋ”‡†Ž‹‡Ȍ‘”‰Ǧ…‡ŽŽ•ȋ„Ž—‡Ž‹‡Ȍˆ‘”„‘—†ƒ–‹„‘†›ʹ†ƒ›•
ˆ‘ŽŽ‘™‹‰ –”ƒ•ˆ‡” ‹–‘ ‹—‹œ‡† ”‡…‹’‹‡–• ƒ† ’”‹‘” –‘ –”ƒ•ˆ‡” ‹–‘ ‘Ǧ
‹—‹œ‡†‘”‡™Ž›‹—‹œ‡†”‡…‹’‹‡–•Ǥ‡’”‡•‡–ƒ–‹˜‡ˆŽ‘™’Ž‘–•ƒ”‡”‡ˆŽ‡…–‹˜‡
‘ˆ –Š”‡‡ ‹†‡’‡†‡– ‡š’‡”‹‡–•Ǥ ƒ…Š ‡š’‡”‹‡– ”‡’”‡•‡–• ͵ –‘ ͷ ‹…‡ ’‡”
‰”‘—’Ǥ
Supplemental Figure 2 RBC clearance following transfer into non-immunized or
immunized FVB recipients. Ž‡ƒ”ƒ…‡ ‹‡–‹…• ‘ˆ ‰Ϊ • ‹ ‘Ǧ‹—‹œ‡† ‘”
‹—‹œ‡† ”‡…‹’‹‡–• ƒ– ˜ƒ”‹‘—• –‹‡ ’‘‹–• ƒ• ‹†‹…ƒ–‡†Ǥ ƒ–ƒ ƒ”‡
”‡’”‡•‡–ƒ–‹˜‡ ‘ˆ ƒ– Ž‡ƒ•– –Š”‡‡ ‹†‡’‡†‡– ‡š’‡”‹‡–• ƒ† ‡ƒ…Š ‡š’‡”‹‡–
”‡’”‡•‡–•͵–‘ͷ‹…‡’‡”‰”‘—’ƒ–‡ƒ…Š–‹‡’‘‹–ƒƒŽ›œ‡†Ǥ
Supplemental Figure 3 Antigen positive cells undergo antigen modulation and cells
with intermediate levels of antibody exhibit reduced sensitivity to clearance ȋƒ–ƒ
†‹•’Žƒ›‡† ƒ• •–ƒ…‡† Š‹•–‘‰”ƒ•Ǥ ‡‡ ˆ‹‰—”‡• ͷǡ ƒ† ͸Ǧ ˆ‘” …‘’ƒ”‹•‘ȌǤ (A)
Ž‘™ …›–‘‡–”‹… ‡šƒ‹ƒ–‹‘ ‘ˆ ‰ ’‘•‹–‹˜‡ …‡ŽŽ• ‘” ‰ ‡‰ƒ–‹˜‡ …‡ŽŽ• ˆ‘” „‘—†
ƒ–‹„‘†› ˆ‘ŽŽ‘™‹‰ –”ƒ•ˆ‡” ‹–‘ ‹—‹œ‡† ‘” ‘Ǧ‹—‹œ‡† ”‡…‹’‹‡–• ƒ– –Š‡
–‹‡•‹†‹…ƒ–‡†Ǥ(B) Ž‘™…›–‘‡–”‹…‡šƒ‹ƒ–‹‘‘ˆ‰’‘•‹–‹˜‡…‡ŽŽ•‘”‰‡‰ƒ–‹˜‡
…‡ŽŽ• ˆ‘” †‡–‡…–ƒ„Ž‡ ƒ–‹‰‡ ˆ‘ŽŽ‘™‹‰ –”ƒ•ˆ‡” ‹–‘ ‹—‹œ‡† ‘” ‘Ǧ‹—‹œ‡†
”‡…‹’‹‡–•ƒ––Š‡–‹‡’‘‹–•‹†‹…ƒ–‡†Ǥ(C) ‹•–‹…–’‘’—Žƒ–‹‘•‘ˆƒ–‹‰‡Š‹‰ŠȏŠ‹Ȑ
‘”ƒ–‹‰‡‹–‡”‡†‹ƒ–‡ȏ‹–Ȑ…‡ŽŽ•–”ƒ•ˆ‡””‡†‹–‘‘Ǧ‹—‹œ‡†”‡…‹’‹‡–•Ǥ(D)
šƒ‹ƒ–‹‘‘ˆƒ–‹„‘†›„‘—†‘”Ž‡˜‡Ž•‘ˆ†‡–‡…–ƒ„Ž‡ƒ–‹‰‡‘–”ƒ•ˆ‡””‡†…‡ŽŽ•
™‹–Š ȏͲȐǡ ȏ‹–Ȑǡ ‘” ȏŠ‹Ȑ…‘…‡–”ƒ–‹‘• ‘ˆ ƒ–‹Ǧ›͵ ƒ– –Š‡ ‹†‹…ƒ–‡† –‹‡’‘‹–• ‘ ‰
’‘•‹–‹˜‡ …‡ŽŽ• ‘” ‰ ‡‰ƒ–‹˜‡ …‡ŽŽ•Ǥ ‡’”‡•‡–ƒ–‹˜‡ ˆŽ‘™’Ž‘–• ƒ”‡ ”‡ˆŽ‡…–‹˜‡ ‘ˆ–Š”‡‡
‹†‡’‡†‡– ‡š’‡”‹‡–•Ǥ ƒ…Š ‡š’‡”‹‡– ”‡’”‡•‡–• ͵ –‘ ͷ ‹…‡ ’‡” ‰”‘—’ ƒ–
‡ƒ…Š–‹‡’‘‹–ƒƒŽ›œ‡†Ǥ
Supplemental Figure 4 Antigen modulation occurs following transfer of Ag positive
cells into immunized FVB recipients. šƒ‹ƒ–‹‘ ‘ˆ „‘—† ƒ–‹„‘†› ‘” †‡–‡…–ƒ„Ž‡
ƒ–‹‰‡ ʹ †ƒ›• ˆ‘ŽŽ‘™‹‰ –”ƒ•ˆ‡” ‘ˆ ‰Ϊ …‡ŽŽ• ȋ•‘Ž‹† „Žƒ… Ž‹‡Ȍ ‘” ‰Ǧ …‡ŽŽ• ȋ‰”ƒ›Ȍ
‹–‘ ‘Ǧ‹—‹œ‡† ‘” ‹—‹œ‡† ”‡…‹’‹‡–•Ǥ ‡’”‡•‡–ƒ–‹˜‡ ˆŽ‘™ ’Ž‘–• ƒ”‡
”‡ˆŽ‡…–‹˜‡ ‘ˆ –Š”‡‡ ‹†‡’‡†‡– ‡š’‡”‹‡–•Ǥ ƒ…Š ‡š’‡”‹‡– ”‡’”‡•‡–• ͵ –‘ ͷ
‹…‡’‡”‰”‘—’ƒ–‡ƒ…Š–‹‡’‘‹–ƒƒŽ›œ‡†Ǥ
Supplemental Figure 1
Supplemental Figure 2
Supplemental Figure 3
Supplemental Figure 4