Functional CD47/signal regulatory protein alpha
(SIRPα) interaction is required for optimal human
T- and natural killer- (NK) cell homeostasis in vivo
Nicolas Legranda,b,1, Nicholas D. Huntingtonc,d, Maho Nagasawaa, Arjen Q. Bakkerb, Remko Schottea,e,
Hélène Strick-Marchandc,d, Sandra J. de Geusf, Stephan M. Pouwa, Martino Böhnea,b, Arie Voordouwa,
Kees Weijera, James P. Di Santoc,d, and Hergen Spitsa,b,g,1
aDepartment of Cell Biology and Histology, Academic Medical Center of the University of Amsterdam (AMC-UvA), Center for Immunology Amsterdam (CIA),
1105AZ, Amsterdam, The Netherlands;bAIMM Therapeutics, 1105BA, Amsterdam, The Netherlands;cInnate Immunity Unit, Institut Pasteur, 75724 Paris,
France;dInstitut National de la Santé et de la Recherche Médicale U668, Institut Pasteur, 75724 Paris, France;eDivision of Immunology, The Netherlands
Cancer Institute, 1183NG, Amsterdam, The Netherlands;fDepartment of Pathology, Vrije Universiteit Medical Center, 1081HV, Amsterdam, The Netherlands;
andgTytgat Institute for Liver and Intestinal Research, Academic Medical Center of the University of Amsterdam (AMC-UvA), 1105BK, Amsterdam, The
Edited* by Richard A. Flavell, Yale University School of Medicine, Howard Hughes Medical Institute, New Haven, CT, and approved June 14, 2011 (received for
review January 26, 2011)
The homeostatic control mechanisms regulating human leukocyte
numbers are poorly understood. Here, we assessed the role of
phagocytes in this process using human immune system (HIS)
BALB/c Rag2−/−IL-2Rγc−/−mice in which human leukocytes are gen-
erated from transplanted hematopoietic progenitor cells. Interac-
tions between signal regulatory protein alpha (SIRPα; expressed on
phagocytes) and CD47 (expressed on hematopoietic cells) nega-
tively regulate phagocyte activity of macrophages and other
phagocytic cells. We previously showed that B cells develop and
survive robustly in HIS mice, whereas T and natural killer (NK) cells
survive poorly. Because human CD47 does not interact with BALB/c
mouse SIRPα, we introduced functional CD47/SIRPα interactions in
HIS mice by transducing mouse CD47 into human progenitor cells.
Here, we show that this procedure resulted in a dramatic and se-
lective improvement of progenitor cell engraftment and human
T- and NK-cell homeostasis in HIS mouse peripheral lymphoid
organs. The amount of engrafted human B cells also increased
but much less than that of T and NK cells, and total plasma IgM
and IgG concentrations increased 68- and 35-fold, respectively.
Whereas T cells exhibit an activated/memory phenotype in the
absence of functional CD47/SIRPα interactions, human T cells accu-
mulated as CD4+or CD8+single-positive, naive, resting T cells in the
presence of functional CD47/SIRPα interactions. Thus, in addition to
signals mediated by T cell receptor (TCR)/MHC and/or IL/IL receptor
interactions, sensing of cell surface CD47 expression by phagocyte
SIRPα is a critical determinant of T- and NK-cell homeostasis under
steady-state conditions in vivo.
hematopoiesis|humanized mice|leukocyte homeostasis
actions with both hematopoietic cells and nonhematopoietic
components. The dependency of lymphocytes on such signals has
been intensely studied in mice, and several factors regulating
lymphocyte numbers in steady-state conditions have been iden-
tified (1–3). Because of ethical and practical reasons, experi-
mental exploration of human leukocyte homeostatic mechanisms
in vivo is difficult. To address this topic, mouse models with
components of the human immune system (HIS) have been
generated by injecting human hematopoietic progenitor cells
(hHPC) into newborn immunodeficient mice (4–6).
HIS mice generated in BALB/c Rag2−/−IL-2Rγc−/−newborns
support multilineage human hematopoietic development, but
human T- and natural killer- (NK) cell homeostasis remains sub-
optimal in this model (7, 8). Signs of disturbed human T-cell ho-
meostasis include premature thymus aging, low T-cell frequency
in peripheral lymphoid organs, high peripheral T-cell turnover,
and high variability in naive T-cell proportion (7–9). HIS mice
omeostasis of hematopoietic cells depends on molecular
signals provided by extracellular factors and cell–cell inter-
complemented with human IL-7 (10–12) or human MHC mole-
cules (13) (i.e., two major factors controlling naive T-cell ho-
meostasis) exhibit enhanced human T-cell numbers but only to
a limited extent. Similarly, inoculation of human IL-15/IL-15Rα
into HIS mice leads to improved but still suboptimal NK-cell ac-
cumulation(14).HIS micegenerated in adult BALB/cRag2−/−IL-
2Rγc−/−mice, which contain more phagocytes than newborn ani-
mals, only show limited accumulation of human B cells (15). In
contrast, HIS mice generated in immunodeficient mice based on
the nonobese diabetic (NOD) genetic background, known for its
defective phagocyte activity (4, 16), exhibit a large proportion of
human T and NK cells in their lymphoid organs (10, 17, 18).
plays a role in T- and NK-cell number regulation.
Phagocyte activity is inhibited by interactions between CD47
and signal regulatory protein alpha (SIRPα; CD172a) (19). CD47,
a ubiquitously expressed Ig-like membrane protein, can interact
with several other cell surface receptors (e.g., thrombospondin,
integrins, and other members of the SIRP family) (20). These
receptors can interact with CD47 in cis or in trans, and these
interactions are described as a two-way exchange of information
(21). The resulting complexity of CD47 biology may explain its
broad—but not fully elucidated—role in hematopoietic and im-
mune processes. The expression pattern of SIRPα is restricted to
neuronal and hematopoietic lineages, with a remarkable bias to
immune cells exhibiting a phagocytic activity (e.g., macrophages,
granulocytes, or dendritic cells) (19). CD47nullmice are viable and
apparently healthy; however, phenotypic differences with their
CD47+littermates are revealed in a pathogenic setting, because
CD47−/−mice are unable to control Escherichia coli bacterial in-
fection (22). CD47-deficient erythrocytes injected into CD47+
mice are rapidly cleared from the blood circulation by phagocytes
of the recipient, suggesting that CD47 ligation is a critical dis-
criminator of self (23). Similarly, experimental settings making
to CD47−/−leukocytes is only observed in absence of CD47 ex-
pression on nonhematopoietic cells (24). These observations
highlight that CD47/SIRPα interactions are a major determinant
of escape from phagocyte-mediated cell clearance.
Author contributions: N.L., N.D.H., J.P.D.S., and H.S. designed research; N.L., N.D.H., R.S.,
H.S.-M., S.J.d.G., S.M.P., M.B., A.V., and K.W. performed research; N.D.H., M.N., A.Q.B.,
A.V., K.W., and J.P.D.S. contributed new reagents/analytic tools; N.L., N.D.H., R.S., H.S.-M.,
M.B., J.P.D.S., and H.S. analyzed data; and N.L., J.P.D.S., and H.S. wrote the paper.
The authors declare no conflict of interest.
*This Direct Submission article had a prearranged editor.
1To whom correspondence may be addressed. E-mail: firstname.lastname@example.org or hergen.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
| August 9, 2011
| vol. 108
| no. 32 www.pnas.org/cgi/doi/10.1073/pnas.1101398108
Based on this set of observations, we investigated in HIS mice
whether CD47/SIRPα interactions would function as a mecha-
nism of immune surveillance regulating human leukocyte num-
bers in lymphoid organs. It was previously shown that human
hematopoietic reconstitution of HIS mice is improved when
proper CD47/SIRPα interactions take place (25), but the relative
in vivo impact on the diverse human leukocyte subsets was not
analyzed. Here, we show that in vivo homeostasis of human T and
NK cells is particularly sensitive to sensing of correct CD47 ex-
pression on cell surface (i.e., phagocytes selectively participate to
their homeostatic control in steady-state conditions).
Enforced Expression of Mouse CD47 by Human Cells Improves Human
Xeno-Engraftment in Vivo. To assess the role of CD47 in human
hematopoietic cell maintenance in vivo, we made use of a human-
ized mouse model of the hemato-lymphoid cellular components
(4–6). We and others have already shown that transplantation
of hHPC into BALB/c Rag2−/−IL-2Rγc−/−newborn mice leads to
multilineage human hematopoietic reconstitution (7, 8, 14). The
human cells found in BALB-HIS mice cohabit with components
of the mouse immune system (e.g., macrophages and dendritic
cells). Most laboratory mouse strains, including BALB/c, express
an allele of the SIRPα molecule that does not properly bind to
human CD47 (25, 26). We, therefore, tested whether human he-
matopoietic cells with enforced mouse CD47 (mCD47) expression
would exhibit improved survival in the BALB-HIS mice. Before
the mCD47-expressing pHEF lentiviral vector (Fig. S1A). In
mCD47/BALB-HIS mice, mCD47 expression on human cells was
restricted to the transduced cells (Fig. S1B).
Twelve weeks after mCD47-hHPC transplantation, the fre-
quency (Fig. 1A and Fig. S2A) and number (Fig. 1B and Fig. S2B)
of human cells harvested from mCD47/BALB-HIS mice were
enhanced in all lymphoid organs (2.5- to 7.5-fold more). We
compared the proportion of GFP+cells obtained in vitro on
hHPC after transduction and in the reconstituted HIS mice (Fig.
S2C). The improved human graft maintenance correlated with
a selective advantage for mCD47-expressing human cells (Fig.
S2D). No GFP+cell accumulation was observed in the control
group (27). We next analyzed the relative contribution of GFP−
and GFP+cells to human hematopoiesis in mCD47/BALB-HIS
hHPC generated a similar amount of human cells, indicating that
the lentiviral transduction did not induce any bias (27). In con-
trast, GFP+hHPC expressing mCD47 systematically generated
higher numbers of human cells (7.3- to 27.5-fold more) (Fig. 1C).
Human hematopoietic reconstitution of C57BL/6 Rag2−/−IL-
of achievement is potentially because of the high activity of
the murine myeloid cells in this genetic background (4). To test
whether mCD47-enforced expression can bypass this apparent
roadblock, we generated mCD47/B6-HIS mice with newborn
C57BL/6 Rag2−/−IL-2Rγc−/−animals, and we observed a dramatic
enhancement of human cell repopulation in all lymphoid organs
(Fig. S3). The level of human cell accumulation in the mCD47/B6-
previously described as nonpermissive to hHPC engraftment.
Enforced mCD47 Expression Results in Improved Engraftment of hHPC
in Vivo. The improvement in human engraftment in mCD47/
BALB-HIS mice could be caused by two nonmutually exclusive
mechanisms: (i) the number of multipotent hHPC might be in-
creased in these animals, therefore giving rise to enhanced num-
be conferred a specific survival advantage. To test the first possi-
2+) (Fig. S4A) hHPC engraftment capacity and long-term main-
tenance was enhanced by mCD47 expression. The frequency of
hHPC was increased approximately fourfold in GFP+human cells
hHPC recovered from control animals was similar in both GFP−
weeks 1 and 4 after transplantation and no more accumulation
after week 4 (Fig. 2B). A positive effect of mCD47-enforced ex-
pression on hHPC numbers was already detectable asearlyas 1 wk
posttransplantation, with a 9.3-fold increase in GFP+hHPC
recovery (Fig. 2B). The number of hHPC recovered from the
mCD47-expressing GFP+cells was accumulating over time (∼30-
fold between weeks 1 and 4 posttransplantation and ∼5-fold be-
tween weeks 4 and 12).
Overall, enforced expression of mCD47 by human cells
improves short- and long-term human hHPC engraftment in
Enforced mCD47 Expression in hHPC Leads to Improved Human
Thymopoiesis. We next investigated whether specific hematopoi-
etic lineages were exhibiting a selective survival advantage when
expressing mCD47. We focused on T and NK cells, which are
described to poorly accumulate in BALB-HIS mice (5, 6, 9). In
the thymus, the frequencies of pro-T cells (CD1a+CD3−) and
CD4+CD8+double-positive (DP) immature thymocytes subsets
were increased in mCD47/BALB-HIS mice, whereas the fre-
quencies of CD4+(SP4), CD8+(SP8) single-positive thymo-
cytes, and mature thymocytes (CD1a−CD3+) were reduced (Fig.
S4 B–D). Enhanced thymopoiesis in mCD47/BALB-HIS mice
hHPC. Human cell engraftment in 12-wk-old control (n = 9) and mCD47/
BALB-HIS mice (n = 10). (A) Frequency and (B) total number of human cells
harvested from BALB-HIS mice generated either with control- or mCD47-
transduced hHPC (horizontal bar is the mean value). (C) Number of hCD45+
cells generated in vivo from a normalized amount (10,000) of GFP−or GFP+
hHPC (mean + SEM). When statistically significant, the increase in cell
numbers observed in mCD47/BALB-HIS mice is indicated (fold increase over
control). BM, bone marrow.
Human cell engraftment in BALB-HIS mice using mCD47-expressing
Legrand et al. PNAS
| August 9, 2011
| vol. 108
| no. 32
was mostly caused by the specific accumulation of mCD47-
expressing cells, because mCD47-expressing hHPC generated
more pro-T cells (10.4-fold), immature DP thymocytes (6.9-
fold), and mature T cells (10.6-fold) than their control GFP+
counterparts (Fig. S4E). The proportions of CD16−CD56+and
CD16hiCD56lowthymic NK-cell subsets were similar between the
two groups of animals (Fig. S4F). Because mCD47-transduced
hHPC generated more human thymocytes (7.3-fold increase)
(Fig. 1C), the total number of thymic NKp46+NK cells gener-
ated from mCD47-hHPC was enhanced in a similar proportion
(5.3-fold increase) (Fig. S4F).
Altogether, these results show that proper CD47 ligation on hu-
increase of thymus size, more physiological proportions of major
thymocyte subsets, and enhanced numbers of immature thymocytes.
mCD47-Expressing T and NK Cells Selectively Accumulate in BALB-HIS
in peripheral lymphoid organs. We consistently observed a pref-
erential increase of T-cell frequency within mCD47/BALB-HIS
GFP+cells (∼16% of hCD45+GFP+splenocytes vs. ∼5% in
control animals), whereas B-cell frequency was not significantly
different between the two groups, resulting in a 5.9-fold reduction
expressing hHPC fractiongenerated ∼45-foldmore matureT cells
inthespleencomparedwithcontrol ornontransducedhHPC (Fig.
3B). Similar selective accumulation of T cells was observed in all
analyzed lymphoid organs (bone marrow, liver, and blood). The
number of B and myeloid cells generated by GFP+hHPC wasalso
increased in mCD47/BALB-HIS mouse spleens but to a lower
extent (B cells: 15.8-fold increase; monocytes: 14.3-fold increase)
compared with T cells (Fig. 3B and Fig. S5B). Histological analysis
of the spleen of the mCD47/BALB-HIS mice revealed an im-
proved organization of human T cells, which formed clusters to-
gether with B-cell areas, whereas T cells were more randomly
scattered inside the spleen of control animals (Fig. 3C). The fre-
animals), mostly because of the accumulation of CD16hi-
CD56loNKp46+NK cells (∼6.6% of hCD45+CD3−GFP+sple-
nocytes vs. ∼1.1% in control animals, P = 0.0149) (Fig. 3D).
Overall, mCD47-expressing hHPC generated ∼97-fold more NK
cells in the spleen (Fig. 3E).
Altogether, these data show that, whereas mCD47 expression
induced an accumulation of all leukocyte subsets in BALB-HIS
mice, there was a selective increase of peripheral T- and NK-cell
numbers, resulting in the reversal of the human B- to T-cell ratio.
Frequency of CD34+CD38−hHPC among human GFP+cells harvested in the
bone marrow of 12-wk-old control (n = 9) and mCD47/BALB-HIS (n = 10)
mice. (B) Normalized number of CD34+CD38−hHPC recovered at week 1
(liver; n = 5 control vs. n = 5 mCD47), week 4 (bone marrow; n = 8 control vs.
n = 7 mCD47), and week 12 (bone marrow; n = 9 control vs. n = 10 mCD47)
Kinetic of hHPC niche reconstitution in mCD47/BALB-HIS mice. (A)
HIS mice (n = 10). (A) Representative CD3 and CD19 expression on hCD45+GFP+splenocytes harvested from 12-wk-old mice. Graphs show frequency of T cells
(CD3+) and B- to T-cell ratio among GFP+human splenocytes. (B) Normalized number of human T and B cells (from 10,000 hHPC). (C) Histological analysis of
the spleen for the presence of human hematopoietic cells (hCD45), T (hCD3), and B cells (hCD20). H/E, H&E. Pictures were obtained in successive sections from
one representative animal from each group. (D) Representative CD16 and CD56 expression among hCD45+GFP+CD3−splenocytes and NKp46 expression
among GFP+CD16hiCD56loNK cells. The graph shows the frequency of total NK cells (pooled CD16−CD56+NKp46+and CD16hiCD56loNKp46+) among
hCD45+GFP+cells. (E) Normalized number of human total NK cells (from 10,000 hHPC).
Accumulation of mCD47-expressing T, B, and NK cells in the spleen. Human T-, B-, and NK-cell subsets in the spleen of control (n = 8) and mCD47/BALB-
| www.pnas.org/cgi/doi/10.1073/pnas.1101398108Legrand et al.
mCD47-Expressing T Cells Accumulate in BALB-HIS Mice as Naïve,
Resting Cells and Exhibit Improved Survival Capacity. We next
monitored the relative proportion of several T-cell subsets. The
frequency of CD4+and CD8+T cells in the spleen did not sig-
nificantly differ between groups, but mCD47 expression was ac-
T cells (Fig. 4A), which may correspond to activated T cells nor-
mally found at low frequency in healthy individuals but that accu-
mulate in inflammatory conditions (28). The mCD47-expressing
hHPC generated ∼43-fold more CD4+T cells, ∼68-fold more
counterparts (Fig. 4B). Using CCR7 and CD45RA cell surface
markers (29), we observed that mCD47-expressing mature CD4+
and CD8+T lymphocytes accumulated in lymphoid organs mostly
as naive T cells (TN; CD45RA+CCR7+), whereas T cells mostly
belonged to the central memory T-cell (TCM; CD45RA−CCR7+)
T cells (TEM; CD45RA−CCR7−) did not significantly differ be-
tween the two groups (Fig. 4C). Furthermore, cycling (Ki67+) T
in the spleen of mCD47/BALB-HIS mice, whereas a significant
fraction of control GFP+(∼65%) or nontransduced T cells
(∼30%) were actively dividing (Fig. 4D) (9).
We assessed the functionality of human leukocyte subsets
generated in mCD47/BALB-HIS mice. Improved T-cell ho-
meostasis was accompanied by a marked accumulation of total
plasma IgM and IgG in mCD47/BALB-HIS mice, with 68- and
35-fold increases, respectively, compared with control mice (Fig.
4E). We next tested the capacity of mCD47-expressing T cells to
survive in lymphopenic-recipient mice after adoptive transfer.
We inoculated human cells isolated from mCD47/BALB-HIS
mice into nonmanipulated BALB/c Rag2−/−IL-2Rγc−/−mice
(i.e., the same host environment). One day after mouse T-cell
adoptive transfer, the recovery in the spleen typically represents
1–10% of the original inoculum (30). When injecting control
BALB-HIS T cells, the D+1 recovery in the spleen was very
limited (∼0.08% of the inoculum), and the cells became un-
detectable at D+3 (Fig. 4F). The initial recovery of mCD47-
expressing T cells was 15-fold better (∼1.2% of the inoculum),
and these T cells were maintained at a similar level for at least 8
d (Fig. 4F). The maintenance of mCD47-expressing T cells was
accompanied by moderate T-cell division (Fig. S6A) and no overt
phenotypic change over time (Fig. S6B). Culture of mCD47/
BALB-HIS spleen NK cells in the presence of K562 human
leukemia cells induced the surface expression of CD107a, a de-
granulation marker, at a level that was higher (Fig. S6C) or
similar (14) to what was observed with control cells. Last, mCD47/
BALB-HIS spleen T cells cultured with allogeneic human pe-
ripheral blood mononuclear cells extensively proliferated to
a similar extent as control cells (Fig. S6D).
From these results, we conclude that proper CD47 ligation
supports optimal T- and NK-cell homeostasis and their re-
spective functional activities.
Engraftment of hHPC in BALB/c NOD.sirpa Rag2−/−IL-2Rγc−/−Mice. To
confirm that CD47/SIRPα interactions are responsible for the
aforementioned observations, we generated BALB/c Rag2−/−IL-
2Rγc−/−mice congenic for the NOD.sirpa gene, which interacts
with human CD47 (25). Similar to what we observed in mCD47/
BALB-HIS mice, human cells harvested from NOD.sirpa-HIS mice
were increased 2.0- to 7.5-fold (Fig. 5A). The hHPC pool repre-
sented ∼3% of bone marrow human cells compared with ∼1% in
control animals, and the number of hHPC was approximately
eightfold higherin NOD-sirpa congenic mice(Fig.S7A).SplenicT
cells accumulated ∼22-fold more in NOD.sirpa-HIS mice than in
control animals, resulting in an 7.9-fold reduction in the B- to T-
cellratio (Fig.5B).Asa comparison,B-cell number increasedonly
2.7-fold (control: 6.1 ± 0.2 × 106vs. NOD.sirpa: 16.6 ± 6.9 × 106,
P= 0.1251).NOD.sirpa-HISperipheralTcellsmostly belongedto
the TNcell subset (Fig. 5C). Furthermore, 11.2-fold more human
5D and Fig. S7B). Although only rare mesenteric lymph nodes
could be harvested from control BALB-HIS mice, various en-
larged lymph nodes (e.g., mesenteric, brachial, axillary, or in-
guinal) could be isolated from NOD.sirpa-HIS mice, resulting
in approximately fivefold higher human cell numbers (Fig. S7C).
control (n = 9) or mCD47/BALB-HIS (n = 10) mice. (B) Normalized numbers of CD4+, CD8+, and DP T cells (from 10,000 hHPC). (C) Proportion of TN, TCM, and TEM
cells in the CD4+, CD8+, and DP GFP+T cells. (D) Isotype control and Ki67 staining on splenocytes from one representative mouse from each group. The
percentage of Ki67+T cells is given. The numerical analysis is shown in the graph (n = 5 per group). (E) Plasma concentration of total human IgM and IgG in
12-wk-old control (n = 9) vs. mCD47/BALB-HIS (n = 27) mice (IgM: 1.1 ± 0.4 vs. 77.8 ± 25.6 μg/mL; IgG: 15.3 ± 7.9 vs. 529.1 ± 112.6 μg/mL). (F) Adoptive transfer
of control or mCD47/BALB-HIS spleen T cells into adult, nonmanipulated BALB/c Rag2−/−IL-2Rγc−/−mice. The number of human GFP+T cells recovered from the
spleen of the recipient mice is plotted as a percentage of recovery from the initial inoculum (n = 4 control vs. n = 3 mCD47 per time point).
Phenotype and survival capacity of mCD47-expressing T cells. (A) Proportion of CD4+, CD8+, and DP cells among GFP+spleen T cells in 12-wk-old
Legrand et al.PNAS
| August 9, 2011
| vol. 108
| no. 32
T-cell proportion was also increased in the lymph nodes of NOD.
sirpa-HIS mice, resulting in a 7.3-fold reduction in the B- to T-cell
ratio (Fig. S7C). Overall, NOD.sirpa-HIS mice recapitulated the
observations obtained in BALB-HIS mice, including a selective
enhancement of human T- and NK-cell reconstitution.
In this study, we have used mice humanized for components of
axis as a major mechanism controlling human T- and NK-cell ho-
meostasis. By using two complementary experimental approaches,
weshow that CD47 is the major—if not the only—ligand of SIRPα
in this setting. By ensuring proper ligation of SIRPα (mouse
phagocytes) by CD47 (human cells), human hematopoietic cell
maintenance was globally improved, in conjunction with selective
accumulation of human T and NK cells (i.e., two subsets showing
poor accumulation in the BALB-HIS mouse model).
There are several explanations for the improved maintenance of
human cells in the presence of functional CD47/SIRPα inter-
actions. First, sustained increase in hHPC numbers likely leads to
increased numbers of their hematopoietic offspring. Second, hu-
man hematopoietic cells expressing mCD47 exhibited a marked
competitive advantage during hematopoietic reconstitution. Third,
in most lymphoid organs analyzed, we also observed a benefit
of mCD47-enforced expression on the maintenance of non-
transduced hematopoietic cells of the same hosts. This observation
suggests that mCD47+human cells induced partial mouse phago-
cyte tolerance to mCD47−cells. In mice, inoculation of CD47null
hematopoietic cells into CD47+mice results in their rapid clear-
or NK cell-depleted animals (23, 31), whereas splenectomy or
clodronate-mediated phagocyte depletion significantly reduces
their elimination (23). In (WT→CD47KO) bone marrow chimeras
generated with WT mouse progenitor cells and lethally irradiated
CD47−/−animals, infused CD47−/−splenocytes were shown to
survive for at least 3 d (24). In contrast, CD47−/−splenocytes
injected either in (WT→WT) or (CD47KO→WT) bone marrow
concluded that the lack of CD47 expression on nonhematopoietic
cells induces phagocyte tolerance to CD47nullleukocytes, which
would normally beeliminated in thespleen. In BALB-HIS mice,in
which nonhematopoietic cells are mCD47+hCD47null, mouse
phagocytes may function as mediators of immunosurveillance (32)
and actively limit the engraftment of mCD47nullhHPC. In-
troduction of optimal CD47/SIRPα interactions enables the de-
livery of proper do not eat me signals to mouse phagocytes by
human hematopoietic cells, even in limiting conditions such as in
newborn C57BL/6 (Fig. S3) or macrophage-sufficient BALB/c
adult mice (Fig. S8) or after injection of low hHPC numbers (Fig.
HIS (from GFP+hHPC) (Fig. 3), NOD.sirpa-HIS (Fig. 5), and
NSG-HIS mice (Fig. S9) were of the same order of magnitude.
Our data strongly suggest that human T-cell homeostasis is
particularly sensitive to the CD47/SIRPα signaling axis. In the
thymus, mCD47−human cells did not benefit from induction of
mouse phagocyte tolerance by mCD47+cells. In peripheral
lymphoid organs, effective CD47/SIRPα interactions lead to en-
hanced B-cell and monocyte generation (∼15-fold increase in
spleen) but to a limited extent compared with T cells (∼45-fold).
As a consequence, mCD47+T cells selectively accumulate in
peripheral lymphoid organs, mostly as naive, resting cells, with
improved function, which was evidenced by increased total
plasma IgM and IgG concentration. Until the present report,
CD47 role in T-cell development and peripheral survival
remained particularly elusive, although evidences suggested that
T cells were sensitive to CD47-mediated clearance mechanisms
in vivo. In CD47−/−mice, a limited, nonsignificant reduction in
peripheral T-cell frequency was reported (22). It was later shown
that thymocyte numbers are reduced approximately twofold in
CD47−/−mice (33). Phagocytes in CD47−/−mice are tolerant to
other CD47nullhematopoietic cells, and therefore, experiments
have been designed to test the behavior of CD47−/−bone marrow
cells injected in sublethally irradiated CD47+/+hosts (i.e., during
competitive reconstitution). In this experimental setup, a com-
plete lack of de novo CD47−/−T-cell generation was observed
over a 1-y period, whereas CD47−/−B and myeloid cells were
detected for up to 25 and 35 wk after reconstitution, respectively
(24). It, therefore, seems that T-cell lineage is particularly sensi-
tive to phagocyte-mediated removal.
Our data highlight two other noticeable consequences of
functional CD47/SIRPα interactions, namely improved NK-cell
homeostasis and lymph node organogenesis. Similarly to T cells,
selective accumulation of NK cells was obtained in presence of
proper CD47/SIRPα interactions. Of note, this specific effect was
observed at the periphery but not in the thymus of humanized
mice, suggesting that homeostasis of thymic and peripheral NK-
cell subsets might be differentially regulated (2, 34). Improved
lymph node organogenesis was also systematically observed both
in mCD47/BALB-HIS and NOD.sirpa-HIS mice. In contrast,
control BALB-HIS mice only exhibited rare mesenteric lymph
nodes (7, 8). It is known that lymphoid tissue inducer cells are
strongly reduced in IL-2Rγc−/−mice because of lack of IL-7 sig-
naling (35). Adult Rag2−/−IL-2Rγc−/−mice lack Peyer’s patches
and exhibit rudimental anlagen of peripheral lymph nodes (4–6).
Still, these anlagen are normally generated during embryonic life
Human cell repopulation incontrol(n=6)and NOD.sirpa-HISmice(n =5)12wk
after hHPC injection. (A) Total number of human cells in the bone marrow,
thymus, and spleen. (B) T-cell frequency, total T-cell number, and B- to T-cell
CD4+and CD8+T cells. (D) Number of human NK cells (NKp46+) in the spleen.
Human cell reconstitution in BALB/c NOD.sirpa Rag2−/−IL-2Rγc−/−mice.
| www.pnas.org/cgi/doi/10.1073/pnas.1101398108Legrand et al.
and can be maintain during neonatal life in an IL-7/IL-7R–de-
pendent fashion by colonizing hematopoietic cells (e.g., through
the adoptive transfer of T or NK cells but not B cells within 1 wk
of birth) (36). In HIS mice, proper CD47/SIRPα interactions may
allow for the rapid development of human cells, supporting
maturation of Rag2−/−IL-2Rγc−/−mouse lymph node anlages.
Indeed, we observed, in mCD47/BALB-HIS mice, low but sig-
nificant numbers of Lin−CD34−CD117+CD127+cells, which
might represent human lymphoid tissue inducer cells (37).
Phagocytes exert well-described functions in the clearance of
apoptotic cells and aged erythrocytes (38, 39) as well as immu-
nosurveillance mediators of tumor cells and virus-infected cells
(20, 32). Our results provide a role for CD47-dependent phago-
cyte-mediated mechanisms for the control of hematopoietic cell
numbers. Next to MHC/T cell receptor (TCR) (13) and IL-7/IL-
7R interactions (10–12) for T cells and IL-15/IL-15R interactions
forNKcells (14),we,therefore,propose CD47/SIRPαinteraction
as another critical mechanism regulating human T- and NK-cell
homeostasis under steady-state conditions. In humans, such
a mechanism could be driven by differential expression of CD47
between various hematopoietic cell subsets, which was proposed
in the case of senescent erythrocytes (38). The question of
whether CD47 expression during an infection or in certain path-
specific human hematopoietic cell populations still remains open.
Materials and Methods
Details are in SI Materials and Methods.
mCD47/BALB-HIS Mouse Generation and Analysis. HIS mice were generated as
previously described (7, 8, 27). Fetal liver CD34+CD38−lineage-negative (CD3,
CD11c, CD19, CD56, and BDCA2) hHPC were sorted using a FACS-Aria (BD
Biosciences). Newborn (<5 d old) sublethally irradiated (3.5 Gy) BALB/c
Rag2−/−IL-2Rγc−/−mice were injected intrahepatic with ∼105hHPC, trans-
duced either with a control or codon-optimized mCD47-expressing pHEF
lentiviral vector (11, 27). BALB/c Rag2−/−IL-2Rγc−/−NOD.sirpa congenic ani-
mals were generated, bred, and maintained at the Institut Pasteur (by N.D.H
and J.P.D.S.) after backcrossing to BALB/c background for six generations.
Cell suspensions were stained with fluorescent anti-human mAbs targeting
the indicated cell markers and analyzed with an LSR-II cytometer (BD Bio-
sciences). Dead cells were excluded based on DAPI incorporation.
Histology and ELISA. Histological analysis was performed on formaldehyde-
fixed, paraffin-embedded tissue samples. Stainings were performed on
successive sections either with H&E or monoclonal antibodies to hCD45 (LCA
2B11+PD7/26; Dako), hCD20 (L26; Dako), or hCD3 (SP7; Neomarkers). The
plasma samples were screened by ELISA for the presence of total human IgM
[AffiniPure F(ab’)2goat anti-hIgM and IgG AffiniPure goat anti-hIgG; Jack-
son ImmunoResearch). Plasma samples were tested in serial dilutions starting
at a 1:2 dilution.
Human Cell Adoptive Transfers. HIS mouse spleen cell suspensions were
injected i.v. into adult (>10 wk old) nonirradiated BALB/c Rag2−/−IL-2Rγc−/−
mice. Each recipient mouse received 1.5–3.5 × 106hCD45+cells. To correct for
the variable frequency of GFP+T cells in the original inoculum, the number of
human T cells recovered from the recipient mice is plotted as a percentage of
recovery from the inoculum. In some experiments, the human cells were la-
beled with the cell division tracking dye CellTrace-Violet (15 μM; Invitrogen).
ACKNOWLEDGMENTS. We thank Dr. Timo van den Berg and Dr. Mireille
Centlivre for their valuable suggestions, Berend Hooibrink for expert
maintenance of the flow cytometry platform, the Bloemenhove Clinic
(Heemstede, The Netherlands) for providing fetal tissues, and the staff of
the Animal Research Institute Amsterdam for animal care. This work was
supported by grants from the Bill and Melinda Gates Foundation (Grand
Challenges in Global Health Program GC4), Wijnand M. Pon Foundation,
Collège de France, Fondation pour la Recherche Médicale (FRM), Institut
Pasteur, and Institut National de la Santé et de la Recherche Médicale
(INSERM). N.D.H. was supported by the Human Frontiers Science Program,
and R.S. was supported by Dutch Cancer Society Grant NKI 2006-3530.
1. Almeida AR, Rocha B, Freitas AA, Tanchot C (2005) Homeostasis of T cell numbers:
From thymus production to peripheral compartmentalization and the indexation of
regulatory T cells. Semin Immunol 17:239–249.
2. Huntington ND, Vosshenrich CA, Di Santo JP (2007) Developmental pathways that
generate natural-killer-cell diversity in mice and humans. Nat Rev Immunol 7:703–714.
3. Takada K, Jameson SC (2009) Naive T cell homeostasis: From awareness of space to
a sense of place. Nat Rev Immunol 9:823–832.
4. Legrand N, Weijer K, Spits H (2006) Experimental models to study development and
function of the human immune system in vivo. J Immunol 176:2053–2058.
5. Manz MG (2007) Human-hemato-lymphoid-system mice: Opportunities and chal-
lenges. Immunity 26:537–541.
6. Shultz LD, Ishikawa F, Greiner DL (2007) Humanized mice in translational biomedical
research. Nat Rev Immunol 7:118–130.
7. Gimeno R, et al. (2004) Monitoring the effect of gene silencing by RNA interference in
human CD34+ cells injected into newborn RAG2-/- gammac-/- mice: Functional in-
activation of p53 in developing T cells. Blood 104:3886–3893.
8. Traggiai E, et al. (2004) Development of a human adaptive immune system in cord
blood cell-transplanted mice. Science 304:104–107.
9. Legrand N, et al. (2006) Transient accumulation of human mature thymocytes and
regulatory T cells with CD28 superagonist in “human immune system” Rag2(-/-)
gammac(-/-) mice. Blood 108:238–245.
10. Shultz LD, et al. (2005) Human lymphoid and myeloid cell development in NOD/LtSz-
scid IL2R gamma null mice engrafted with mobilized human hemopoietic stem cells.
J Immunol 174:6477–6489.
11. van Lent AU, et al. (2009) IL-7 enhances thymic human T cell development in “human
immune system” Rag2-/-IL-2Rgammac-/- mice without affecting peripheral T cell ho-
meostasis. J Immunol 183:7645–7655.
12. O’Connell RM, et al. (2010) Lentiviral vector delivery of human interleukin-7 (hIL-7) to
13. Manz MG, Di Santo JP (2009) Renaissance for mouse models of human hematopoiesis
and immunobiology. Nat Immunol 10:1039–1042.
14. Huntington ND, et al. (2009) IL-15 trans-presentation promotes human NK cell de-
velopment and differentiation in vivo. J Exp Med 206:25–34.
15. Weijer K, et al. (2002) Intrathymic and extrathymic development of human plasma-
cytoid dendritic cell precursors in vivo. Blood 99:2752–2759.
16. Shultz LD, et al. (1995) Multiple defects in innate and adaptive immunologic function
in NOD/LtSz-scid mice. J Immunol 154:180–191.
17. Ito M, et al. (2002) NOD/SCID/gamma(c)(null) mouse: An excellent recipient mouse
model for engraftment of human cells. Blood 100:3175–3182.
18. Ishikawa F, et al. (2005) Development of functional human blood and immune sys-
tems in NOD/SCID/IL2 receptor gamma chain(null) mice. Blood 106:1565–1573.
19. Barclay AN (2009) Signal regulatory protein alpha (SIRPα)/CD47 interaction and
function. Curr Opin Immunol 21:47–52.
20. van Beek EM, Cochrane F, Barclay AN, van den Berg TK (2005) Signal regulatory
proteins in the immune system. J Immunol 175:7781–7787.
21. Sarfati M, Fortin G, Raymond M, Susin S (2008) CD47 in the immune response: Role of
thrombospondin and SIRP-alpha reverse signaling. Curr Drug Targets 9:842–850.
22. Lindberg FP, et al. (1996) Decreased resistance to bacterial infection and granulocyte
defects in IAP-deficient mice. Science 274:795–798.
23. Oldenborg PA, et al. (2000) Role of CD47 as a marker of self on red blood cells. Science
24. Wang H, et al. (2007) Lack of CD47 on nonhematopoietic cells induces split macro-
phage tolerance to CD47null cells. Proc Natl Acad Sci USA 104:13744–13749.
25. Takenaka K, et al. (2007) Polymorphism in Sirpα modulates engraftment of human
hematopoietic stem cells. Nat Immunol 8:1313–1323.
26. Subramanian S, Parthasarathy R, Sen S, Boder ET, Discher DE (2006) Species- and cell
type-specific interactions between CD47 and human SIRPα. Blood 107:2548–2556.
in “human immune system” Rag2-/- gamma c -/- mice. Methods Mol Biol 595:87–115.
28. Parel Y, Chizzolini C (2004) CD4+ CD8+ double positive (DP) T cells in health and
disease. Autoimmun Rev 3:215–220.
29. Sallusto F, Lenig D, Förster R, Lipp M, Lanzavecchia A (1999) Two subsets of memory T
lymphocytes with distinct homing potentials and effector functions. Nature 401:708–712.
30. Hao Y, Legrand N, Freitas AA (2006) The clone size of peripheral CD8 T cells is reg-
ulated by TCR promiscuity. J Exp Med 203:1643–1649.
31. Blazar BR, et al. (2001) CD47 (integrin-associated protein) engagement of dendritic
cell and macrophage counterreceptors is required to prevent the clearance of donor
lymphohematopoietic cells. J Exp Med 194:541–549.
32. Jaiswal S, Chao MP, Majeti R, Weissman IL (2010) Macrophages as mediators of tumor
immunosurveillance. Trends Immunol 31:212–219.
33. Guimont-Desrochers F, et al. (2009) Absence of CD47 in vivo influences thymic dendritic
34. VosshenrichCA,etal. (2006)Athymicpathwayofmousenaturalkillercelldevelopment
characterized by expression of GATA-3 and CD127. Nat Immunol 7:1217–1224.
35. Cupedo T, Mebius RE (2005) Cellular interactions in lymph node development. J Im-
36. Coles MC, et al. (2006) Role of T and NK cells and IL7/IL7r interactions during neonatal
maturation of lymph nodes. Proc Natl Acad Sci USA 103:13457–13462.
37. Cupedo T, et al. (2009) Human fetal lymphoid tissue-inducer cells are interleukin 17-
producing precursors to RORC+ CD127+ natural killer-like cells. Nat Immunol 10:66–74.
38. Oldenborg PA (2004) Role of CD47 in erythroid cells and in autoimmunity. Leuk
39. Gardai SJ, et al. (2005) Cell-surface calreticulin initiates clearance of viable or apo-
ptotic cells through trans-activation of LRP on the phagocyte. Cell 123:321–334.
Legrand et al.PNAS
| August 9, 2011
| vol. 108
| no. 32
Legrand et al. 10.1073/pnas.1101398108
SI Materials and Methods
Human Tissues, Cell Preparation, and Lentiviral Transductions. Hu-
man fetal livers were obtained from elective abortions with
approved by the Medical Ethical Committee of the Academic
Medical Center of the University of Amsterdam and was con-
tingent on informed consent. Fetal cells were prepared as pre-
viously described (1). The CD34+CD38−lineage-negative (CD3,
CD11c, CD19, CD56, or BDCA2) human hematopoietic pro-
genitor cells (hHPC) were sorted using a FACS-Aria interfaced to
a FACS-Diva software system (BD Biosciences). The cDNA se-
quence encoding a codon-optimized mouse (m) CD47 (GeneArt)
was inserted into the multiple cloning site of the self-inactivated
third generation lentiviral pHEF vector (2), downstream of the
human EF1α promoter and upstream of an internal ribosomal
entry site and enhanced GFP (1). The control vector was an
empty pHEF internal ribosomal entry site-GFP vector. Lentiviral
supernatants were produced on 293T cells as previously described
(1). Lentivirus stocks were concentrated with ultra-concentrator
columns (Amicon), aliquoted, and stored at −80 °C before
transduction of hHPC. The hHPC were cultured overnight in
Iscove’s modified Dulbecco’s medium (Invitrogen) supplemented
with Yssel’s medium (3), 5% normal human serum, 20 ng/mL
human thrombopoietin (PeproTech), stem cell factor (Pepro-
Tech), hIL-7 (Cytheris), and hIL-3 (PeproTech). The following
day, cells were incubated for 6–8 h with lentivirus supernatant
in fibronectin-coated plates (30 μg/mL; Takara), washed, and
injected. An aliquot was maintained in culture for 4 d before
measuring the frequency of GFP+cells.
Flow Cytometry Analysis. Cell suspensions were stained with FITC,
phycoerythrin (PE), PerCP-Cy5.5, PE-Cy7, allophycocyanin
(APC), APC-Cy7, or Alexa-Fluor 700 labeled anti-human mAb
targeting the following cell surface markers: CD1a (T6-RD1) and
CD38 (CLT16) from Beckman Coulter; CD3 (SK7), CD4 (SK3),
CD8 (SK1), CD11c (B-ly6), CD14 (M5E2), CD16 (3G8), CD19
(HIB19), CD34 (581), CD38 (HIT2), CD45 (2D1 and HI30), CD47
(HCD47), CD45RA (HI100), CD56 (B159), CD107a (H4A3),
CD117 (YB5.B8), natural killer (NK)p46 (9E2), and CCR7 (3D12)
from BD Biosciences and BioLegend; Ki67 from BD Biosciences;
cells were excluded based on DAPI incorporation. All washings and
reagent dilutions were performed with PBS containing 2% FCS and
0.02% NaN3. Stained cells were analyzed with an LSR-II cytometer
interfaced to a FACS-Diva software system (BD Biosciences).
Animals. BALB/c Rag2−/−IL-2Rγc−/−(BALB) (4), C57BL/6 Ra-
g2−/−IL-2Rγc−/−(B6; Charles River), and nonobese diabetic
(NOD) .Cg-PrkdcscidIL-2RgtmlWjl/Sz (Jackson Laboratory) mice
were bred and maintained in individual ventilated cages at the
Animal Research Institute Amsterdam and fed with autoclaved
food and water. BALB/c Rag2−/−IL-2Rγc−/−NOD.sirpa congenic
animals were generated, bred, and maintained at the Institut Pas-
teur (by N.D.H. and J.P.D.S.). These mice were generated by
crossing the NOD.Cg-Prkdcscidmice with BALB/c Rag2−/−IL-
2Rγc−/−and backcrossing NOD.sirpa+offspring with BALB/c
Rag2−/−IL-2Rγc−/−mice for six generations. The expression of the
forward5′-3′GCC ACG AGG ACA GAA GTG A;reverse5′-
3′ AAG TCC AAG TTA CTT CTC TTA GTA GCA.
Generation of Human Immune System Mice. BALB-human immune
system(HIS) mice were generated as previously described (1,5, 6)
with the approval of the Animal Ethical Committee of the Aca-
demic Medical Center of the University of Amsterdam and the
Institut Pasteur. Newborn (<5 d old), sublethally irradiated (3.5
Gy) BALB/c Rag2−/−IL-2Rγc−/−mice were injected through in-
trahepatic route with ∼1 × 105transduced hHPC. B6-HIS and
same procedure. NSG-HIS mice were produced with a reduced
irradiation dose (1 Gy). Adult-BALB-HIS mice were produced by
mice were performed under laminar flow. The HIS mice were
exsanguinated under isofluran/oxygen narcosis, and the blood was
harvested in the presence of EDTA (∼4.5 mM final concentra-
tion). Pictures were taken using iPhone 3GS (Apple Inc.).
Histology and ELISA. Histological analysis was performed on form-
aldehyde-fixed, paraffin-embedded tissue samples. Sections were
cut at 3 μm, and immunochemistry stainings were performed on
successive sections using LabVision Autostainer 480S (Thermo
Scientific) either with H&E or commercially available monoclonal
antibodies to hCD45 (LCA 2B11+PD7/26; Dako), hCD20 (L26;
by ELISA for the presence of total human IgM and IgG by coating
96-well plates either with AffiniPure F(ab’)2fragment goat anti-
human IgM (Fc5μ-specific) or AffiniPure goat anti-human IgG
(Fcγ-specific; both from Jackson ImmunoResearch). Control hu-
man serumprotein calibrator(Dako) was used asstandardforIgM
(0.8 mg/mL)andIgG (10.4mg/mL). Plasmasamples weretestedin
serial dilutions, starting at a dilution of 1:2. ELISA was revealed
with HRP-conjugated anti-human IgM or anti-human IgG anti-
bodies (Jackson ImmunoResearch) used at 1:2,500 dilution.
Human Leukocyte Functional Assays. For adoptive transfers, leuko-
cytes were isolated from spleens by performing a density gradient
injected i.v. into adult (>10 wk old), nonirradiated BALB/c Ra-
g2−/−IL-2Rγc−/−mice. Each recipient mouse received 1.5–3.5 × 106
in the original inoculum, the number of human T cells recovered
from the recipient mice is plotted as a percentage of recovery from
the inoculum. NK-cell stimulation was performed by culturing total
× 106cells/mL). T-cell alloreactivity was assessed by culturing total
of rhIL-2 (50 U/mL; Roche) with or without 40-Gy irradiated allo-
geneic human peripheral blood mononuclear cells (mixed donors;
2.5 × 106cells/mL). Where indicated, the human cells were labeled
with the cell division tracking dye CellTrace-Violet (15 μM; In-
vitrogen) using the manufacturer’s instructions.
Normalization Method and Statistical Analysis. To correct for the
different amounts of GFP+vs. GFP−hHPC injected into the
animals, the number of hCD45+cells (and subsets) generated in
vivo was normalized for a fixed amount (10,000) of GFP−or
GFP+hHPC when indicated. Statistical analyses were performed
using GraphPad Prism version 5.0c for Mac (GraphPad Soft-
ware). Data were subjected to two-tailed unpaired Student t test
analysis. The obtained P values were considered significant when
P < 0.05 and were indicated as follows: *P < 0.05; **P < 0.01;
***P < 0.001.
Legrand et al. www.pnas.org/cgi/content/short/1101398108 1 of 7
1. van Lent AU, et al. (2010) In vivo modulation of gene expression by lentiviral
transduction in “human immune system” Rag2-/- gamma c -/- mice. Methods Mol Biol
2. van Lent AU, et al. (2009) IL-7 enhances thymic human T cell development in “human
immune system” Rag2-/-IL-2Rgammac-/- mice without affecting peripheral T cell
homeostasis. J Immunol 183:7645–7655.
3. Yssel H, De Vries JE, Koken M, Van Blitterswijk W, Spits H (1984) Serum-free medium
for generation and propagation of functional human cytotoxic and helper T cell
clones. J Immunol Methods 72:219–227.
4. Weijer K, et al. (2002) Intrathymic and extrathymic development of human
plasmacytoid dendritic cell precursors in vivo. Blood 99:2752–2759.
5. Traggiai E, et al. (2004) Development of a human adaptive immune system in cord
blood cell-transplanted mice. Science 304:104–107.
6. Gimeno R, et al. (2004) Monitoring the effect of gene silencing by RNA interference in
human CD34+ cells injected into newborn RAG2-/- gammac-/- mice: Functional
inactivation of p53 in developing T cells. Blood 104:3886–3893.
(hCD45−) cells harvested from the bone marrow of a representative mCD47/BALB-HIS mouse (dot plot) and GFP expression (histograms) for the following
populations: mouse cells (hCD45−mCD47+), mCD47−human cells, and mCD47+human cells.
Lentiviral vectors. (A) Schematic representation of the pHEF lentiviral vector. (B) Surface expression of mCD47 on human (hCD45+) and mouse
10; data pooled from two independent experiments). (A) Frequency and (B) total number of human cells harvested from liver and blood of 12-wk-old BALB-HIS
mice generated either with control- or mCD47-transduced hHPC. Blood cell numbers are given per milliliter of blood. (C) In vitro hHPC transduction efficiency
and in vivo recovery of GFP+cells (hCD45+gated) in the spleen of 12-wk-old control (Left) and mCD47/BALB-HIS mice (Right). (D) Ratio between the in vivo
frequency of GFP+cells (among hCD45+human cells) and in vitro transduction efficiency.
Human cell engraftment in BALB-HIS mice using mCD47-expressing hHPC. Human cell engraftment in control (n = 9) and mCD47/BALB-HIS mice (n =
Legrand et al. www.pnas.org/cgi/content/short/11013981082 of 7
generated using C57BL/6 Rag2−/−IL-2Rγc−/−newborn mice as hHPC recipients. (A) Frequency (Upper) and the total number (Lower) of hCD45+cells recovered in
lymphoid organs 12 wk posttransplantation. (B) Normalized number of human cells generated from 10,000 hHPC.
Human cell engraftment in B6-HIS mice using mCD47-expressing hHPC. Human cell engraftment in control (n = 13) and mCD47/B6-HIS mice (n = 12)
Legrand et al. www.pnas.org/cgi/content/short/11013981083 of 7
bone marrow cells of representative 12-wk-old control (Upper) and mCD47/BALB-HIS mice (Lower). CD117 and CD133/2 surface expression is shown on gated
CD34+CD38−cells (Right). (B) Human T-cell development in control (n = 8) and mCD47/BALB-HIS mice (n = 10). Representative examples of the CD4 and CD8
expression patterns on hCD45+GFP+thymocytes harvested from 12-wk-old mice. The graph shows the frequency of double-positive cells among GFP+human
thymocytes. (C) Similar analysis for CD1a and CD3 surface expression. Numerical analysis is provided for the proportion of pro-T cells (CD1a+CD3−). (D) The
graphs show the frequency of SP4, SP8 (Upper), immature thymocytes (CD1a+CD3+), and thymus mature T (CD1a−CD3+) cells (Lower) among GFP+human
thymocytes. (E) Normalized number of human pro-T cells, double-positive thymocytes, and thymus mature T cells (from 10,000 hHPC). (F) Representative
examples of the CD16 and CD56 expression among hCD45+GFP+CD3−thymocytes (dot plots). The graphs show the frequency of total NK cells (pooled
CD16−CD56+NKp46+and CD16hiCD56loNKp46+) among hCD45+GFP+cells (Left) and the normalized number of human total thymus NK cells generated from
10,000 hHPC (Right).
Bone marrow hHPC and thymus cell populations in mCD47/BALB-HIS mice. (A) Pattern of CD34 and CD38 surface expression (Left) on the GFP+human
Legrand et al. www.pnas.org/cgi/content/short/1101398108 4 of 7
control (n = 8) and mCD47/BALB-HIS mice (n = 10). (A) Frequency of B cells (CD19+) among GFP+human splenocytes. (B) Frequency of monocytes (CD11c+CD14+)
among hCD45+GFP+cells (Left) and normalized number of human monocytes generated from 10,000 hHPC (Right).
Accumulation of mCD47-expressing human B cells and monocytes in the spleen. Human B-cell and monocyte accumulation in the spleen of 12-wk-old
Rag2−/−IL-2Rγc−/−mice. The phenotype of the transferred T cells was monitored in time (n = 4 control vs. n = 3 mCD47 per time point). The transferred human
splenocytes were labeled with the CellTrace-Violet fluorescent dye before inoculation, and cell division was monitored at days 1, 3, and 8 posttransfer by
following the dilution of the dye among dividing cells (solid line). The gray areas at days 3 ad 8 correspond to the fluorescence pattern at days 1 and 3,
respectively. (B) Expression pattern of CD4, CD8 (Left), CCR7, and CD45RA (Right) on the surface of the transferred GFP+T cells. (C) NK-cell degranulation assay
assessed by the induction of CD107a expression on the surface of control (Upper) and mCD47/BALB-HIS (Lower) spleen NK cells when cocultured for 18 h with
K562 human leukemia cells. The percentage of CD107a+cells among CD56+human cells is indicated (one representative experiment of two experiments). (D) T-
cell alloreactivity was evaluated after 7 d of culture of control (Upper) and mCD47/BALB-HIS (Lower) splenocytes labeled with the CellTrace-Violet fluorescent
dye in the presence or absence of irradiated human peripheral blood mononuclear cells. T-cell division was monitored by the dilution of the dye over time, and
the percentage of CD3+T cells negative for CellTrace-Violet is given (one representative experiment of two experiments).
mCD47/BALB-HIS T- and NK-cell functionality. (A) Adoptive transfer of control or mCD47/BALB-HIS spleen T cells into adult, nonmanipulated BALB/c
Legrand et al. www.pnas.org/cgi/content/short/11013981085 of 7
wk after hHPC injection. (A) Frequency (Left) and total number (Right) of hHPC harvested from the bone marrow. (B) Frequency of human NK cells (NKp46+) in
the spleen. (C) Total number of human cells harvested from pooled lymph nodes (Left) and B- to T-cell ratio in these samples (Center). Enlarged brachial and
axillary lymph nodes observed in NOD.sirpa-HIS mice are indicated by the white arrows on the macroscopic view of one representative animal (Right).
Human cell reconstitution in BALB/c NOD.sirpa Rag2−/−IL-2Rγc−/−mice. Human cell repopulation in control (n = 6) and NOD.sirpa-HIS mice (n = 5) 12
mice (n = 10) generated using BALB/c Rag2−/−IL-2Rγc−/−adult mice as hHPC recipients. (A) Frequency (Upper) and the total number (Lower) of hCD45+cells
recovered in lymphoid organs 12 wk posttransplantation. (B) Normalized number of human cells generated from 10,000 hHPC.
Human cell engraftment in adult BALB-HIS mice using mCD47-expressing hHPC. Human cell engraftment in control (n = 8) and mCD47/Adult-BALB-HIS
Legrand et al. www.pnas.org/cgi/content/short/11013981086 of 7
control or the mCD47-expressing vector and were kept in culture for 36 h before the transduced (GFP+) fraction was sorted. A limited number (10,000) of GFP+-
sorted hHPC was then injected into the newborn mice. (A) Human cell engraftment in control (n = 3) and mCD47/BALB-HIS mice (n = 2) generated using BALB/c
Rag2−/−IL-2Rγc−/−newborn mice as hHPC recipients. Frequency (Upper) and the total number (Lower) of hCD45+cells recovered in lymphoid organs 12 wk
posttransplantation. (B) A similar analysis was performed on control (n = 4) and mCD47/NSG-HIS mice (n = 4) generated using NSG newborn mice as hHPC
recipients. (C) Normalized number of human cells generated from 10,000 hHPC. (D) Frequency and normalized numbers (generated from 10,000 hHPC) of
spleen T (Upper) and NK cells (Lower) from each group. The normalized cell numbers can be directly compared with the data shown in Fig. 3 B and E. To
compare these data with what we obtained in NOD.sirpa-HIS mice, total numbers of T and NK cells shown in Fig. 5 should be divided by a factor of 10 to
normalize for 10,000 hHPC. ND, not detected.
Human cell engraftment in BALB-HIS and NSG-HIS mice using limited numbers of mCD47-expressing hHPC. hHPC were either transduced with the
Legrand et al. www.pnas.org/cgi/content/short/11013981087 of 7