macrophages prevent human red blood cell reconstitution in

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Macrophages prevent human red blood cell reconstitution in immunodeficient mice

Zheng Hu1,2,3, Nico Van Rooijen4, Yong-Guang Yang2,3

1 Institute of Immunology, Hefei National Laboratory for Physical Sciences at Microscale and

School of Life Sciences, University of Science and Technology of China, Hefei, China

2 Transplantation Biology Research Center, Massachusetts General Hospital, Harvard Medical

School, Boston, MA

3 Columbia Center for Translational Immunology, Columbia University College of Physicians

and Surgeons, New York, NY

4 Department of Cell Biology, Vrije Universiteit, Amsterdam, The Netherlands

Short title: Human red blood cell reconstitution in mice

Corresponding Author: Yong-Guang Yang, MD, PhD

Columbia Center for Translational Immunology, Columbia University College of Physicians and

Surgeons, 650 West 168th St, BB1501, New York, NY 10032

Tel (212) 304-5586; Fax (646) 426-0036; Email [email protected]

Blood First Edition Paper, prepublished online September 16, 2011; DOI 10.1182/blood-2010-11-321414

Copyright © 2011 American Society of Hematology

For personal use only.on April 10, 2019. by guest www.bloodjournal.orgFrom

http://www.bloodjournal.org/

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2

Abstract

An animal model supporting human erythropoiesis will be highly valuable for assessing the

biological function of human RBCs under physiological and disease settings, and for evaluating

protocols of in vitro RBC differentiation. Herein, we analyzed human RBC reconstitution in

NOD/SCID or NOD/SCID/γc-/- mice that were transplanted with human CD34+ fetal liver cells

and fetal thymic tissue. Although a large number of human CD45-CD71+ nucleated immature

erythroid cells were detected in the bone marrow, human RBCs were undetectable in the blood

of these mice. Human RBCs became detectable in blood following macrophage depletion, but

disappeared again after withdrawal of treatment. Furthermore, treatment with human

erythropoietin and IL-3 significantly increased human RBC reconstitution in macrophage-

depleted, but not control, humanized mice. Significantly more rapid rejection of human RBCs

than CD47-deficient mouse RBCs indicates that mechanisms other than insufficient CD47-

SIRPα signaling are involved in human RBC xenorejection in mice. All considered, our data

demonstrate that human RBCs are highly susceptible to rejection by macrophages in

immunodeficient mice. Thus, strategies for preventing human RBC rejection by macrophages

are required for using immunodeficient mice as an in vivo model to study human erythropoiesis

and RBC function.




3

Introduction

Red blood cells are part of non-immune blood-lineage cells derived from hematopoietic stem

cells. Anemia continues to exert tremendous burdens to human health.1-4 Thus, a considerable

effort has been made to develop new therapies for the treatment of anemia. Although the focus in

the treatment of anemia has been on growth factors (e.g., erythropoietin, EPO), derivation of

hematopoietic cells from human embryonic stem cells (hESCs) and induced pluripotent stem

cells (iPSCs) are considered a worthwhile endeavor in the areas of transfusion therapies.5 It has

been reported that enucleated RBCs can be differentiated from hESCs with functional oxygen-

carrying ability on large scale.6 However, these studies have been limited to in vitro assays due

to the lack of a suitable animal model.

Immunodeficient mice provide a very useful in vivo model for the study of human

lymphohematopoietic cell function. Human hematopoietic stem cell engraftment and

differentiation can now be reproducibly established in NOD/SCID mice or their derivatives.7

However, it remains unclear if immunodeficient mice are suitable for the study of human RBC

differentiation and function. Although a previous report showed detectable human RBCs in

NOD/SCID/IL2rnull (NSG) mice receiving human cord blood CD34+ cell transplantation via the

facial vein at the newborn stage,8 the ability of immunodeficient mice to support human

erythropoiesis and/or survival of human RBCs has not been confirmed by other studies.9

Recently, hydrodynamic injection of pcDNA-encoding EPO and IL-3 was found to improve

human RBC reconstitution in NSG mice receiving CD34+ cells, but the levels of human RBCs in

these mice were extremely low compared to the levels of human PBMCs.10




4

We have previously developed a humanized mouse model, in which co-transplantation of human

fetal thymic tissue (under renal capsule) and CD34+ fetal liver cells (FLCs; i.v.) resulted in

sustained repopulation with multilineages of human lymphohematopoietic cells, including T, B

and dendritic cells, and the formation of secondary lymphoid organs in NOD/SCID mice.11-13 In

this study, we assessed human RBC reconstitution in these humanized mice. We observed that

human RBCs were undetectable in blood circulation, despite the fact that large numbers of

human normoblasts were detected in the bone marrow. Furthermore, the lack of human RBCs in

blood circulation was largely accounted for by the extremely high susceptibility of human RBCs

to rejection by recipient mouse macrophages.

Materials and Methods

Animals and human tissues and cells

NOD.CB17-Prkdcscid/J (NOD/SCID), NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NOD/SCID/γc-/- or

NSG), and B6.129-Itgptm1Fpl (CD47 KO B6) mice were purchased from the Jackson Laboratory

(Bar Harbor, ME), and were housed in a specific pathogen-free micro-isolator environment and

used in experiments at 6 to 10 weeks of age. GFP-transgenic CD47 KO mice were generated by

crossing CD47 KO mice with C57BL/6-transgenic (UBC-GFP)30Scha/J (The Jackson

Laboratory) mice. Human fetal thymus and liver tissues of gestational age of 17 to 20 weeks

were obtained from Advanced Bioscience Resource (Alameda, CA). Protocols involving the use

of human tissues and animals were approved by the Human Research Committee and

Subcommittee on Research Animal Care of the Massachusetts General Hospital (Boston, MA)




5

and Columbia University Medical Center (New York, NY), and all of the experiments were

performed in accordance with the protocols.

Humanized mouse preparation

NOD/SCID or NSG mice were conditioned with sublethal (2.25 Gy) total body irradiation, and

received human CD34+ FLCs (1-5×105/mouse, i.v.) alone, or along with a human fetal thymic

tissue fragment measuring about 1 mm3 (under the recipient kidney capsule) from the same fetal

donor, as previously described 12; 13. CD34+ FLCs were isolated by a magnetic-activated cell

sorter (MACS) separation system using anti-CD34 microbeads (Miltenyi Biotech, Aubum, CA).

Levels of human hematopoietic cells in humanized mice were determined by flow cytometric

(FCM) analysis using various combinations of the following mAbs: anti-human CD45, CD19,

CD3, CD4, CD8, CD71, CD235a; anti-mouse CD45 and Ter119; and isotype control mAbs (all

purchased from BD PharMingen, San Diego, CA). RBCs were collected by tail vein bleeding

and heparin was used for anticoagulation. Mononuclear cells were purified by density gradient

centrifugation with Histopaque 1077 (Sigma-Aldrich, St. Louis, MO). Analysis was performed

on a FACSCalibur or FACSCanto (Becton Dickinson, Mountain View, CA). Dead cells were

excluded from the analysis by gating out lower forward scatter and high propidium iodide-

retaining cells.

Morphological analyses of human erythroid cells from humanized mice

Erythroid cells were purified from blood and bone marrow by cell sorting, suspended in PBS and

centrifuged (800rpm for 5 min) onto glass slides using a Cytospin centrifuge (Shandon; Cheshire,

UK). The slides were stained with the DipQuick Stain Kit (modified Wright Giemsa staining)




6

from Jorgensen Laboratories (Loveland, CO). Stained slides were examined under a Zeiss

microscope and photographed using a Nikon Coolpix 5000 digital color camera.

Macrophage depletion in mice

Macrophage depletion in vivo was performed by intravenous injection of liposome-encapsulated

CL2MDP (dichloromethylene diphosphonate or clodronate). Clodronate was a kind gift of Roche

Diagnostic (Mannheim, Germany) and liposome-encapsulated clodronate (clodronate-liposomes)

was prepared as described.14 Clodronate-liposomes were given at 100 μl per mouse for the 1st

injection, and 50 μl or 100 μl per mouse thereafter, with an interval of 2-7 days as indicated.

Control mice were treated at the same times with liposome-encapsulated PBS (PBS-liposomes).

The efficacy of macrophage depletion was confirmed by measuring the clearance of infused

CD47-/- and CD47+/+ mouse RBCs in randomly selected mice as previously described, 15; 16 and

efficient macrophage depletion is confirmed by the lack of rejection of CD47-/- RBCs. In some

experiments, macrophage depletion was also confirmed by immunohistochemistry using anti-

mouse F4/80 mAb (Figure S1).

Human cytokine treatment

In order to determine whether human erythroid progenitors in humanized mice can respond to

human hematopoietic cytokines, we injected (i.p.) some humanized mice with human EPO (30

U/mouse) and rhIL-3 (100 ng/mouse) once every 3 days throughout the observation period.

Controls were injected i.p. with PBS on the same schedule.

Human RBC clearance assay




7

The human RBC clearance assay was performed as previously described.15 Briefly, fresh human

RBCs were labeled with CFSE and injected intravenously into NOD/SCID mice (1.5×108 RBCs

per mouse) that were treated with PBS-liposomes or clodronate-liposomes (100 μl at day -3, and

50 μl at day -1, with respect to RBC transfusion). To determine the kinetics of human RBC

clearance, 5-μl blood samples were collected at various time points and the levels of surviving

CFSE+ human RBCs were measured by FCM analysis.

Phagocytic assay

For in vitro phagocytic assay, 2x105 peritoneal cells were cultured at 37°C for 2 hours to allow

peritoneal macrophages attach to the plate, nonadherent cells were washed off. Attached

macrophages were then cultured with 1x107 CFSE-labeled RBCs in media containing 4%

NOD/SCID sera for 4 hours at 37°C. After lysis of free RBCs by ACK, the cells were stained with

anti-mouse F4/80-PE and analyzed for phagocytosis by FACS. Because peritoneal macrophages in

naïve mice are poorly phagocytic, NOD/SCID mice were injected i.p. with 2% Bio-Gel

polyacrylamide P 100 (1 mL per mouse; Bio-RAD Laboratories Hercules, CA) to activate

macrophages 4 days prior to peritoneal cell harvest. In vivo phagocytic assay was performed by

injecting CFSE-labeled RBCs (1x108 RBCs/ mouse) into peritoneal cavity of NOD/SCID mice 4

days after treatment with 2% Bio-Gel polyacrylamide P 100. Peritoneal cells were collected 4

hours later, lysed of free RBCs, and stained with anti-mouse F4/80-PE. In both in vitro and in vivo

assays, F4/80 and CFSE double positive (i.e., F4/80+CFSE+) cells are considered macrophages that

have engulfed CFSE-labeled RBCs, and the results are presented as percentages of F4/80+CFSE+

cells in F4/80+ cells.




8

Statistical analysis

The level of significant differences in group means was determined by the student’s t test for

parametric data sets. All statistical analysis was performed using Prism 4 (GraphPad Software,

San Diego, CA). A P value of ≤ .05 was considered significant in all analyses herein.

Results

Humanized mice do not have human RBCs in peripheral blood

Humanized mice were prepared by injection (i.v.) of human CD34+ cells alone or along with

implantation of human fetal thymus under the renal capsule in sublethally-irradiated NOD/SCID

or NSG mice. Blood cells were collected between 9 and 38 weeks after transplantation for

measuring human cell chimerism. Consistent with our previous results,12 all humanized mice

showed high levels (>50% on average) of human cell reconstitution in PBMCs, including human

B cells and T cells (T cells were only detected in humanized mice receiving a fetal thymic graft).

Surprisingly, none of these humanized mice showed detectable human CD235a+ RBCs in the

blood at any time point after transplantation, even in those with >80% of human CD45+ cells in

PBMCs (Figure 1A, B). All RBCs in the blood of these mice stained positive for mouse Ter119.

We have also checked human RBC repopulation in various tissues of humanized mice. Except

the liver in which very few (<0.4%) human RBCs were detected, human RBCs were almost

undetectable in other tissues including lung, kidney and spleen (Supplemental Figure S2). These

results demonstrate that human hematopoietic stem cells are incapable of differentiating into

RBCs and/or human RBCs cannot survive in NOD/SCID or NSG mice.




9

Human erythroid cells with an immature phenotype exist in the bone marrow of

humanized mice

Red blood cells are derived from hematopoietic stem cells in bone marrow and subsequently

migrate out into blood after nuclear extrusion.17 To determine whether a potential defect in

human erythroid lineage differentiation is responsible for the lack of human RBCs in blood, we

analyzed human erythropoiesis in the bone marrow from humanized mice. Unlike that observed

in peripheral blood, a large number of human CD235a+CD45- erythroid cells at a stage of

differentiation beyond erythroblasts (i.e., normoblasts or later),18 were detected in the bone

marrow of all humanized mice examined (Figure 2A). As indicated in Figure 2A, bone marrow

cells can be separated into 3 regions, in which erythroid cells are mainly distributed in R2 and R3,

and R4 contains predominantly granulocytes.18 To determine whether these CD235a+CD45- cells

are immature precursors or mature RBCs, we analyzed the distribution of these cells in the

mature erythrocyte- and immature erythroid precursor-enriched regions, which are presented as

R2 and R3, respectively in Figure 2A.18 Human CD235a+ cells were found mostly in R3, with

only a few cells in R2 (Figure 2A). In contrast, mouse Ter119+ erythroid cells were detected in

both R2 and R3 regions. Consistent with this result, intracellular staining with propidium iodide

revealed the presence of large numbers of nucleated erythroid cells in the bone marrow of these

humanized mice (Supplemental Figure S3). Moreover, the levels of human erythroid chimerism

[huCD235a+/(huCD235a+ plus muTer119+)] in region R3 were comparable to those observed for

human CD45+ cell chimerism [i.e., huCD45+/(huCD45+ plus muCD45+) in the bone marrow

(Figure 2B). These results indicate that human erythroid cell differentiation, at least to the

CD235a+CD45- normoblast stage occurred in the bone marrow of these humanized mice. To

further determine human erythroid cell differentiation in the humanized mice, human CD235a+




10

cells were purified from R2 by cell sorting and analyzed by modified Wright Giemsa staining

after cytospin. As shown in Figure 2C, most human CD235a+ cells in R2 were nucleated cells. In

contrast, most mouse Ter119+ cells in R2 were enucleated cells (Figure 2D). However, the level

of CD71 expression on human CD235a+ cells in R2 were lower than those in R3 (Figure 2E),

suggesting that human CD235a+ cells in R2 are relatively more mature than those in R3. It has

been reported that the level of CD71 (transferring receptor) expression decreases during

erythroid cell differentiation.19-21 Thus, the poor reconstitution with human enucleated mature

RBCs in bone marrow and the lack of human RBCs in blood could be due to the inability of

human erythroid precursors to fully mature, or to poor survival or rejection of mature human

RBCs in humanized mice.

Macrophages mediate rapid rejection of human RBCs in NOD/SCID mice

We next determined whether the lack of human RBCs in peripheral blood of humanized mice

was due to the failure of mature RBCs to survive or to rejection. Because NSG mice do not have

functional T, B or NK cells,22 macrophages are the likely potential effectors to reject human

RBCs. Therefore, we first assessed the ability of recipient macrophages to reject human RBCs by

comparing the survival of normal human RBCs in NOD/SCID mice with or without macrophage

depletion. Macrophage depletion was performed by intravenous injection of clodronate-

liposomes, where mice treated with PBS-liposomes were used as controls. CFSE-labeled human

RBCs were rapidly cleared and became almost undetectable by 2 hours after injection into the

control NOD/SCID mice treated with PBS-liposomes (Figure 3A, B, C). In contrast, human RBC

clearance was prevented by depleting macrophages. These results indicate that macrophages

mediate rapid rejection of human RBCs in NOD/SCID mice.




11

We also measured the phagocytosis of human RBCs by NOD/SCID mouse macrophages. CFSE

labeled human or mouse RBCs were injected into peritoneal cavity of NOD/SCID mice, and

phagocytosis of CFSE-labeled RBCs by macrophages were assessed 4 hours later. As shown in

Figure 3D, recipient mouse F4/80+ macrophages were significantly more efficient in engulfing

human than mouse RBCs. Similar results were obtained from in vitro phagocytic assays, in

which NOD/SCID mouse macrophages were capable of engulfing human, but not mouse RBCs

(Figure 3E). Interestingly, phagocytosis of human RBCs was significantly greater in the cultures

added with untreated NOD/SCID mouse sera than in those containing heat-inactivated

NOD/SCID mouse sera, indicating that mouse complement may facilitate phagocytosis of human

RBCs in NOD/SCID mice. These results provide direct evidence for phagocytosis of human

RBCs by NOD/SCID mouse macrophages.

Human RBC reconstitution in peripheral blood of humanized mice after macrophage

depletion

To further determine whether macrophages are responsible for the lack of human RBCs in

humanized mice, we treated humanized mice with clodronate-liposomes 13 weeks after human

CD34+ cell injection and followed human RBC chimerism in these mice. Consistent with the

experiments described above, all of these mice had high levels of human cell reconstitution

(>40%) in PBMC, but none had detectable human RBC chimerism 12 weeks after CD34+ cell

transplantation (1 week prior to macrophage depletion). Because NOD/SCID mice are highly

sensitive to the toxicity associated with repeated injections of clodronate-liposomes, which is

required for preventing macrophage recovery,23 we stopped the treatment after 3 mice died in the




12

clodronate-liposome-treated group (the last injection was given at day 22). Macrophage

depletion resulted in a marked increase in human RBC chimerism in NOD/SCID mice (Figure 4).

The levels of human RBC chimerism in these mice increased over time to approximately 4% on

average by 3 weeks, but declined again after withdrawal of clodronate-liposome injection. In

contrast, human RBCs remained undetectable in the PBS-liposome control group. Flow

cytometric analysis revealed that most human RBCs detected in blood of macrophage-depleted

humanized mice were CD71- (Figure 4C). Since CD71 expression remains detectable in

immature erythroid cells, including early stage of enucleated reticulocytes, 19-21 the data indicate

that human CD235a+ cells detected in the blood of macrophage-depleted humanized mice are

fully matured RBCs. We also compared the ratios of CD71+ cells in human CD235a+ cells in the

“mature erythrocyte-enriched” R2 region (as defined in Figure 2A) in macrophage-depleted and

control humanized mice. Consistent with the data shown in Figure 2E, in PBS-treated mice, all

most all human CD235a+ cells in R2 were CD71+ immature erythroid cells (Figure 4D).

However, in macrophage-depleted mice, approximately half of the human CD235a+ cells were

mature RBCs, as shown by the loss of CD71 expression (Figure 4D). These results demonstrate

that macrophages pose an important barrier to human RBC reconstitution in humanized mice.

Cytokine treatment promotes the reconstitution of human RBCs in macrophage-depleted

humanized mice

The detection of human RBCs in humanized mice after macrophage depletion indicates that

human hematopoietic stem cells can differentiate into mature RBCs in the NOD/SCID mouse

microenvironment. We next determined whether human erythroid progenitors can respond to

human hematopoietic cytokines in macrophage-depleted humanized mice. Humanized mice with




13

no detectable human RBCs were intraperitoneally injected with PBS (controls) or human

cytokines (EPO and rhIL-3), together with intravenous injection of clodronate-liposomes

(starting on the same day as the cytokine treatment). EPO (30 U/mouse) and rhIL-3 (100

ng/mouse) were injected once every 3 days throughout the observation period. Clodronate-

liposome treatment was discontinued after the last injection at day 25 due to the severe toxicity.

Human RBCs became detectable in blood by 1 week after injection of clodronate-liposomes in

all groups of humanized mice, and there was no significant difference in the levels of human

RBC chimerism between the cytokine-treated and control groups by week 3 (Figure 5). In the 4th

week, the levels of human RBC chimerism increased significantly, but more rapidly in mice

treated with EPO plus IL-3 than that observed for the control group (Figure 5). However, human

RBCs remained undetectable in humanized mice that were treated with human cytokines alone

(Figure 5). These data indicate that human erythroid progenitors developing in humanized mice

are capable of responding to human cytokines. This is consistent with a recent study showing

that bone marrow recovered from humanized mice could differentiate into human erythroid cells

ex vivo 24. The failure of human cytokine treatment to improve human RBC reconstitution in

humanized mice without macrophage depletion further supports the argument that macrophages

pose a critical obstacle to human RBC reconstitution in humanized mice.

Robust rejection of human RBCs in mice can be induced by mechanisms independent of

CD47-SIRPα signaling

The CD47-SIRPα pathway induces inhibitory signals in macrophages and prevents abnormal

phagocytosis of autologous hematopoietic cells.15; 25-27 It has been shown that NOD SIRPα is

capable of cross-reacting with human CD47,28 suggesting that the rejection of human RBCs in




14

humanized mice is unlikely to be predominantly induced by the inability of human CD47 to

interact with the recipient SIRPα. However, our previous studies showed that macrophages in

mixed hematopoietic chimeras, where non-hematopoietic cells do not express CD47, are

tolerized to CD47 KO WBCs, but remain phagocytic against CD47 KO RBCs.16 To determine

the role of the CD47-SIRPα pathway in the rejection of human RBCs in humanized mice, we

compared the rejection rates of human RBCs vs. CD47 KO mouse RBCs in NOD/SCID mice.

Human RBCs were significantly more rapidly cleared than CD47 KO mouse RBCs (Figure 6),

indicating that the rejection of human RBCs in NOD/SCID mice was mainly induced by

mechanisms other than the lack of CD47-SIRPα signaling. In support of this scenario, human

RBCs were also swiftly cleared in T and NK cell-depleted CD47 KO B6 mice, where the lack of

CD47-SIRPα signaling does not trigger phagocytosis (Figure 7). These data suggest that

improving CD47-SIRPα interaction alone is insufficient to prevent human RBC rejection by

mouse macrophages.

Discussion

Since the first humanized SCID (SCID-Hu) mouse model was established over two decades

ago,29 humanized mice have been widely used to study the function of human hematopoietic and

lymphoid cells under normal or disease conditions.7 However, the currently available humanized

mouse models have not been demonstrated to be very useful in assessing human RBC function.

In the study herein, we show that, despite the high levels of human PBMC chimerism, human

mature RBCs were undetectable in NOD/SCID or NSG mice that received transplantation of

CD34+ FLCs or CD34+ FLCs plus human fetal thymic tissues (Figure 1). However, human late-




15

stage immature erythroid cells, i.e., CD235a+CD45- nucleated normoblasts were readily detected

in bone marrow at levels similar to those of human CD45+ cells (Figure 2). Furthermore,

recipient macrophages were found to mediate vigorous rejection of human RBCs (Figure 3), and

macrophage depletion led to detection of human RBCs in the blood circulation of these mice

(Figure 4). Our data demonstrate that overcoming macrophage-mediated rejection is required for

achieving human RBC reconstitution in mice and that other pathway(s) than CD47-SIRPα are

involved in human RBC xenorejection.

A previous report showed that human RBCs remained detectable 3 days after injection of a very

large number (4-5x109/mouse) of cultured human RBCs (cRBCs) into sublethally-irradiated

NOD/SCID mice 21. In this study, the recipient NOD/SCID mice were injected with 4-5x109

“decoy cells” (i.e., human type-O RBCs) 24 hours before cRBC injection. Thus, it is likely that

the short survival of human cRBCs detected in this model was due to saturation of the

phagocytic system by pre-injected “decoy” human type-O RBCs. In support of this possibility,

our data showed that human RBCs remained undetectable in humanized mice after sublethal

irradiation (Supplementary Figure S4).

It remains unclear why only human RBCs but not other hematopoietic cells were rejected by

macrophages in humanized mice. Macrophage activation is regulated by the balance between

activating and inhibitory receptor signals. Previous studies have shown that the CD47-SIRPα

interaction provides inhibitory signal to macrophages and prevents phagocytosis of normal

cells.15; 25-27 Furthermore, the lack of cross-species reactivity between CD47 and SIRPα has

been shown to induce phagocytosis of xenogeneic cells by macrophages.30; 31 Although human




16

CD47 has been shown to cross-react with NOD SIRPα,28 it remains unclear whether human

CD47 is as efficient as mouse CD47 in inducing SIRPα signaling in NOD mouse macrophages,

and if the cross-reaction between human CD47 and NOD SIRPα is sufficient in preventing

human RBCs from phagocytosis by NOD mouse macrophages. Because enucleated RBCs are

more sensitive to phagocytosis than nucleated cells,32; 33 a possible explanation for the rejection

of human RBCs but not nucleated human blood cells could be that the cross-reactivity of human

CD47 with NOD SIRPα is not optimal, which is sufficient for human nucleated hematopoietic

cells, but not RBCs, to prevent rejection by macrophages.

We have previously shown that the lack of CD47 expression on non-hematopoietic cells can

induce split macrophage tolerance in mixed bone marrow chimeras, where macrophages remain

phagocytic against CD47 KO RBCs, but not against nucleated hematopoietic cells, including T,

B and myeloid cells.16 These results indicate that phagocytosis of CD47 KO nucleated

hematopoietic cells, but not RBCs can be prevented by mechanisms independent of the CD47-

SIRPα interaction. Further studies are needed to determine whether such CD47-SIRPα

interaction-independent mechanisms are also involved in the selective protection against

phagocytosis of human nucleated hematopoietic cells in humanized mice. Nonetheless, the

significantly more rapid rejection of human RBCs than CD47 KO mouse RBCs in NOD/SCID

(Figure 6) and CD47 KO (Figure 7) mice indicates that the lack of or insufficient CD47-SIRPα

signaling is unlikely to be the major mechanism triggering macrophage-mediated rejection of

human RBCs in the humanized mice. It is likely that the vigorous rejection of human RBCs by

macrophages is accounted for predominantly by the xenoantigens on human RBCs that can

activate macrophages.34; 35




17

Previous studies have shown the presence of human macrophages and dendritic cells in the

humanized mice.12; 13; 36 However, we do not fully understand why human macrophages did not

reject mouse RBCs in these mice. The fact that non-hematopoietic cells play an important role in

regulating macrophage phagocytic activity and can induce macrophage tolerance 16 raises a

possibility that human macrophages developing in the humanized mice are tolerant of mouse

cells. Further studies are clearly needed to answer these questions.

Although human RBCs became detectable in humanized mice after macrophage depletion, the

levels of human RBC chimerism were still significantly lower than human PBMC chimerism.

Because repeated injections of clodronate-liposomes were highly toxic to NOD/SCID and NSG

mice, we could only treat humanized mice for a short period (approximately 3-4 weeks).

Considering the long lifespan of RBCs (approximately 120 days), it is possible that the limited

recovery in the levels of human RBC chimerism in humanized mice after macrophage depletion

was due to the short period of observation. Even though macrophage-depleted humanized mice

treated with human EPO and IL-3 showed marked increase in the levels of human RBCs in blood

(to 25% on average) at week 4, there was no significant difference in human RBC levels between

these mice and those without cytokine treatment by week 3 (Figure 5). This also suggests that a

longer period of macrophage-depletion will likely permit additional increase in human RBC

chimerism in humanized mice. On the other hand, these data also suggest that the limited human

RBC recovery might be accounted for partially by the relatively poor human erythropoiesis due

to the lack of human hematopoietic growth factors. Furthermore, our data provide evidence that




18

human erythroid progenitors in humanized mice are capable of responding to human EPO and/or

IL-3.

Hematopoietic stem cells differentiate into different lineages of blood cells depending on specific

cytokines, and EPO and IL-3 are essential to erythroid lineage differentiation.21 IL-3 is a

hematopoietic growth factor that plays an important role in hematopoietic stem cell renewal and

differentiation into myeloid, erythroid and megakaryocytic lineages.37-41 IL-3 can be produced by

multiple types of cells, including activated T cells, NK cells, mast cells, and eosinophils.42-44

Mouse cells have been shown to react to human EPO,45 but there is no information indicating

that human cells may also react to mouse EPO in a similar manner. Although human cells do not

react to mouse IL-3,37; 46 it remains unclear if the level of human IL-3 produced by human cells

in humanized mice is sufficient to support human erythropoiesis. Our data indicate that human

IL-3 plus EPO, but not EPO alone (data not shown) could significantly improve human RBC

development in humanized mice (in a period of 4 weeks), suggesting that insufficient human IL-

3 production may contribute to the potentially poor erythropoiesis in humanized mice. However,

the observed large numbers of immature normoblasts in the bone marrow suggest that

humanized mice could support some level of human erythropoiesis. We recently observed that

human megakaryocyte and platelet differentiation are normal in the humanized mice (Hu Z and

Yang YG, unpublished data). Because IL-3 also plays an important role in megakaryocyte and

platelet differentiation,41; 47; 48 these results suggest that the level of human IL-3 in humanized

mice is capable of supporting human hematopoiesis. Thus, although the suboptimal

hematopoietic environment may possibly result in a reduced level of human erythropoiesis in




19

humanized mice, the rejection of mature enucleated RBCs is considered a predominant factor

causing the absence of human RBCs in these mice.

Acknowledgements

The authors thank Drs. Hannes Kalscheuer Hannes and Ben Sprangers for critical review of this

manuscript, and Mr. Orlando Moreno for outstanding animal husbandry. This work was

supported by grants from NIH (RC1 HL100117, PO1 CA111519 and R01 AI064569).

Authorship

Z.H. designed and performed experiments, analyzed data, and wrote the paper; N.VR provided

key reagents; Y-G.Y. conceived the research project, designed experiments, analyzed data, and

wrote the paper.

Conflict-of-interest disclosure: The authors declare no competing financial interest.




20

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26

Figure Legends

Figure 1. Human blood cell reconstitution in humanized mice. NOD/SCID or NSG mice

(n=50) were transplanted with human CD34+ cells alone (n=29) or along with human fetal

thymus tissue (n=21). (A) Levels of human CD45+ cells, CD3+ T cells and CD19+ B cells in

PBMCs, as well as human CD235a+ RBCs in whole blood were analyzed by flow cytometry

between 9 and 38 weeks after human cell/tissue transplantation. Each symbol represents an

individual mouse. (B) Flow cytometry profiles of humanized mouse PBMCs stained with anti-

human CD45 and anti-mouse CD45, and RBCs stained with anti-human CD235a and anti-mouse

Ter119.

Figure 2. Human erythroid cell reconstitution in bone marrow of humanized mice. Bone

marrow cells were harvested from femur and tibia of humanized mice 18 weeks after human

CD34+ FLC transplantation, and analyzed by flow cytometry and cytospin. (A-B) The levels of

huCD45+ and huCD235a+ cells in the indicated regions were determined by flow cytometry.

Shown are flow cytometric profiles (A) and levels (mean± SD; n=9) of human CD45+ cell

chimerism (i.e., % huCD45+ cells in huCD45+ plus muCD45+ cells) and CD235a+ cell chimerism

(i.e., % huCD235a+ cells in huCD235a+ plus Ter119+ cells) in the indicated regions (B). RBCs

were mainly detected in R2 and R3, but not R4 (granulocyte region). *, p<0.05; **, p<0.01; ***

p<0.001; n.s., not significant. (C, D) Human CD235+ cells (C) and mouse Ter119+ cells (D) in

gated R2 region were purified from bone marrow by cell sorting and analyzed morphologically.

The purity (i.e., % of human CD235a or mouse Ter119 cells in the sorted cell population) and

Wright Giemsa staining of the sorted cells are shown in left and right panels, respectively. (E)




27

Representative flow cytometry profiles showing human CD71 expression on gated huCD235a+

cells in R2 and R3. MFI denotes median fluorescence intensity.

Figure 3. Rejection of human RBCs by recipient macrophages in NOD/SCID mice. (A-C)

CFSE-stained human RBCs (1.5×108) were intravenously injected into macrophage depleted

(n=3) or control (n=4) NOD/SCID mice. Blood were collected at the indicated time points and

the levels (%) of injected human RBCs were analyzed by flow cytometry. (A) Representative

flow cytometric profiles. (B) Percentages (mean±SDs) of CFSE+ human RBCs at the indicated

times. (C) Clearance rates of infused human RBCs, i.e., percentages of CFSE+ human RBCs

normalized with the levels at 20 minutes after injection as 100%. (D) CFSE-labeled human or

mouse RBCs (1x108) were injected into peritoneal cavity of NOD/SCID mice, and phagocytosis

of human RBCs (n=3) or mouse RBCs (n=3) by mouse macrophages were measured by staining

with anti-mouse F4/80-PE using flow cytometric analysis. Shown are mean (±SDs) of

phagocytic ratios and representative flow cytometric profiles showing % of CFSE+ cells in the

gated F4/80+ cell population. (E) Human and mouse RBCs were co-cultured with NOD/SCID

mouse macrophages in media containing 4% NOD/SCID mouse sera (untreated or 56oC-heated),

and phagocytosis was measured 4 hours later. Shown are mean (±SDs; n=5 per group) of

phagocytic ratios and flow cytometric profiles showing % of CFSE+ cells in the gated F4/80+ cell

population. Data combined from 2 independent experiments are shown.

Figure 4. Human RBCs reconstitute in humanized mice after macrophage depletion.

Humanized NOD/SCID mice were treated at 13 weeks after human CD34+ FLC transplantation

with clodronate-liposomes (n=6) or PBS-liposomes (n=3) (100 μL at days 0, 2, 7, 12, 17, and 22).




28

In clodronate-liposome treated group, 4 mice died after treatment and numbers of mice available

for analysis were: n=6 for pretreatment; n=4 for weeks 1 and 2; n=3 for weeks 3 and 4; and n=2

for weeks 5 and 6. Blood was collected 1 week prior to and weekly after clodronate-liposome

treatment, and the percentages of human RBCs were determined by flow cytometry. (A)

Representative flow cytometric profiles at week 3 post-treatment. (B) Percentages of human

CD235a+ RBCs in blood at the indicated time points after treatment. Arrow indicates the time we

stopped treatment. (C) Human CD235+ cells were purified by cell sorting from blood of

macrophage-depleted humanized mice, and analyzed by flow cytometry and cytospin. Shown are

flow cytometry profiles of human CD235a and CD71 expression and Wright Giemsa staining of

the purified cells. (D) Percentages (mean±SDs) and representative staining profiles of human

CD71 expression on gated human CD235a+ cells in the “mature erythrocyte-enriched” R2 region

of bone marrow cells (as defined in Figure 2A) from humanized mice treated with PBS-

liposomes (n=3) or clodronate-liposomes (n=3).

Figure 5. Treatment with human EPO and IL-3 promotes human erythropoiesis in

macrophage-depleted humanized mice. Humanized mice were prepared by injection of human

CD34+ FLCs into NOD/SCID mice after sublethal irradiation. At week 13, the mice were treated

with clodronate-liposomes (100μl for the 1st injection and 50μl every 5 days thereafter) plus

human cytokines (EPO and IL-3; n=4) or PBS (n=6), or with human cytokines alone (EPO and

IL-3; n=4). EPO (30U) and rhIL-3 (100 ng) were given every 3 days starting one day after the 1st

injection of clodronate-liposomes. (A) Flow cytometric profiles at week 4 post-treatment. (B)

Percentages (mean±SEMs) of human CD235a+ RBCs in blood at the indicated time points after

treatment. *, p<0.05 for the indicated group compared to the other groups.




29

Figure 6. Human RBCs are more rapidly cleared than CD47 KO mouse RBCs in

NOD/SCID mice. CFSE labeled human RBCs (higher fluorescence) were mixed with CD47

KO-GFP (lower fluorescence) RBCs at 1:1 ratio, and injected i.v. into NOD/SCID mice. Blood

was collected as the indicated times and the levels of surviving human and CD47 KO mouse

RBCs were assessed by flow cytometry. Data shown are representative flow cytometric profiles

(A), levels (%; mean±SEMs) of human and mouse RBCs in blood (B; p<0.0001 for all time

points), and clearance rates of human and mouse RBCs (C; p<0.001). Clearance rates are

presented as the percentages (mean±SEMs) of infused RBCs normalized with the levels at 10

minutes after injection as 100%.

Figure 7. Rapid rejection of human RBCs in CD47KO mice. CD47 KO B6 mice (n=4) were

depleted of T and NK cells by treatment with 1.75mg GK1.5 and 1.4mg 2.43 at day-2, and

150μg PK136 at days -6 and -1, with respect to RBC transfusion. Human and CD47 KO mouse

RBCs were labeled with CFSE at lower and higher concentrations, respectively, mixed at 1:1

ratio, and injected into CD47 KO mice. Blood was collected at the indicated time points for

assessing the levels of human (lower fluorescence) and CD47 KO mouse (higher fluorescence)

RBCs by flow cytometry. (A) Representative flow cytometric profiles at the indicated times. (B)

Level (mean±SDs) of human RBCs or CD47KO RBCs measured at 10 minutes after transfusion.




30

Figure 1

Figure 2

CD45+ CD3+ CD19+ CD235a+0

20

40

60

80

100

Hu

man

cel

ls (

%)

PBMC Blood

muCD45 Ter119

huCD45

huCD235a

A B

99.5%

0%88%

huCD45+

(R1)

huCD235a

+ (R

2)

huCD235a

+ (R3)

0

20

40

60

80

100 ***n.s.

***

Hu

man

cel

ls (%

)A B

FSC huCD45 huCD45

SSC

huCD235a

muCD45

huCD235a

huCD235a

SSC

mTer119 mTer119FSC

R1

R1

R2 R3

R4

R1

R2 R3

C D

R2 huCD235a+ cells

94%

huCD235a

R2 Ter119+ cells

98%

mTer119

EhuCD235a+ cells

R2 R3

huCD71

MFI=1964MFI=365




31

Figure 3

0 100 200 300 400 5000

20

40

60

80

100 PBSClodronate*

***

******

***

Time after transfusion (min)

Cle

aran

ce r

ate

(%)

0 100 200 300 400 5000.00

0.05

0.100.20.30.40.50.6 PBS

Clodronate

***

***

****** ***

***


hu

RB

C in

blo

od

(%)

20 min

40 min

60 min

120 min

240 min

480 min

CFSE

PBS Clodronate

CFSE

A B

C

Human RBC

31.3±6.8

Mouse RBC

7.7±4.3

CFSE0

10

20

30

40

Human Mouse RBC RBC

**

Ph

ago

cytic

rat

io (

%)

0

10

20

30

40

***

*****

RBC Human Human MouseSerum + Heated +

Pha

goc

ytic

rat

io (

%)

D

E

Human RBCs

+ serum

Human RBCs

+ heated

Mouse RBCs

+ serum

CFSE

2.1±0.429.1±3.4 16.8±5.6




32

Figure 4

Figure 5

-1 1 2 3 4 5 6 0.0

1.5

3.0

4.5

6.0

7.5PBSClodronate

Weeks after treatment

Hu

man

RB

Cs

(%)

A B

mTer119

huCD235a

PBS Clodronate

3.360

C

0

20

40

60

80

100 *

PBS Cladronate

% h

uCD

71+ c

ells

inh

uC

D23

5a+

cells

in R

2D

Purified human CD235a+ cells

96% 14%

huCD235a huCD71

PBS Clodronate

huCD71

1 2 3 40

10

20

30

40Clodronate

EPO+IL-3/Clodronate

*EPO+IL-3/PBS

Weeks after treatment

Hu

man

RB

Cs

(%)

B

Ter119

HuCD235a

ClodronateA

6.45

92.32

16.65

80.72

EPO+IL-3/Clodronate

0

99.60

EPO+IL-3/PBS




33

Figure 6

Figure 7

0 100 200 300 400 5000

20

40

60

80

100 Human RBCCD47KO RBC


Cle

aran

ce r

ate

(%)

0 100 200 300 400 5000.0

0.2

0.4

0.6

0.8

1.0 Human RBCCD47KO RBC


Tra

nsf

usi

on

cel

ls (%

)

B C

A

Before 10 min 20 min 40 min 60 min 480 min 4 days

GFP(low) / CFSE(high)

A B

Before

10 min

20 min

40 min

CFSE CD47KO RBC

Human RBC

0.0

0.2

0.4

0.6

0.8

RB

C (

%)

***




doi:10.1182/blood-2010-11-321414Prepublished online September 16, 2011;

Zheng Hu, Nico Van Rooijen and Yong-Guang Yang immunodeficient miceMacrophages prevent human red blood cell reconstitution in

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