Characterization of human B cells in umbilical cord blood-transplanted
Xuefu Wanga, Ziping Qia, Haiming Weia,b, Zhigang Tiana,b, Rui Suna,b,⁎
aInstitute of Immunology, School of Life Sciences, University of Science and Technology of China, Hefei 230027, China
bHefei National Laboratory for Physical Sciences at Microscale, Hefei 230027, China
a b s t r a c t a r t i c l e i n f o
Received 18 September 2011
Received in revised form 5 December 2011
Accepted 7 December 2011
Umbilical cord blood
Hematopoietic stem cells
Human UCB-chimeric mice
Humanized mice are crucially important for preclinical studies. However, the development and potential
function of human B cells in chimeras remain unclear. Here, we describe the study of human B cells in NOD/
LtSzPrkdcscid/J (NOD/SCID) mice. In this study, we transplanted 1.0×105human CD34+cells from umbilical
cord blood (UCB) into NOD/SCID mice after pretreatment with anti-asialo GM1 antiserum and sublethal irradi-
ation. Human CD45+cells were detected in the peripheral blood of the recipient mice from 6 weeks after trans-
plantation.CD19+Bcells accountedfor thegreaterpartoftheCD45+cells inthehumanUCB-chimericmice, but
their maturational stages differed in different organs. Most of the bone marrow (BM) CD19+cells were imma-
ture IgM−IgD−CD24hiCD38hiB cells, whereas the mature CD5+IgM+IgD+CD24intCD38intCD19+B cells were
predominantly present in the spleen and peripheral blood. Human immunoglobulin (Ig) M was detected
in mouse plasma. The human B cells also secreted human interleukin-10 after stimulation with LPS in
vitro. These results show that human CD34+cells can differentiate into human B cells in NOD/SCID mice,
with development and functions that are similar to those of B cell subsets in humans. The transplantation
of human CD34+cells into NOD/SCID mice may provide a useful tool to study the development and function
of human B cells.
© 2011 Elsevier B.V. All rights reserved.
To study the development of human leukocytes and find substi-
tute models for research on vaccines, drug development and autoim-
munity, many efforts have been made to develop human immune
cells in immune-deficient mice over the past two decades [1–5].
Even though advances have been made in generating immune-
deficient mice [6–19], many practical limitations still prevent the hu-
manized mouse models from being used as fully reliable substitutes
for human systems. The function of lymphocytes depends on their de-
normalities in human T cells are partly due to the lack of human MHC
expression in the mouse thymus, which is required for the selection of
T cells [20,21]. Similarly, the function of B cells in humanized mice is
not the same as that in humans. Although discussed in some reports,
the developmental course, subsets and function of B cells in human
UCB-chimeric mice are not very clear [10,22,23].
It was reported that NOD/LtSzPrkdcscid/J (NOD/SCID) mice have
typically been used in the study of the development of human hema-
topoietic stem cells (HSCs) because they have multiple defects in
innate and adaptive immunological function . Additionally, after
treatment with anti-asialo GM1 antiserum, NOD/SCID mice can be ef-
ficiently engrafted with human hematopoietic stem cells [12,13].
Thus, we chose NOD/SCID mice as the recipient mice for the trans-
plantation of human CD34+cells. In humans, bone marrow is the
most important site for B cell development , and B cells from
human peripheral blood lymphocytes can proliferate and secrete an-
tibody in the spleens of immune-deficient mice . However, the
percentages and maturational stages of human B cells developed in
NOD/SCID mice from BM, spleen, peripheral blood and liver are not
Based on the relative expression of CD24 and CD38, B cells can be
divided into three distinct populations: CD19+CD38hiCD24hiB cells
(primarily immature B cells), CD19+CD38intCD24intB cells (primarily
mature B cells), and CD19+CD24hiCD38−B cells (primarily memory
B cells) [27,28]. It was unknown, however, whether the two markers
could also divide human B cells from human UCB-chimeric mice into
similar populations. Additionally, human B cells are divided into B1
and B2 cell subsets, and the expression of CD5 has been used as a
marker of B1a cells, the major subset of B1 cells [29,30]. In humans
and mice, CD5+B1 cells are the major constituent of B cells in the
fetal spleen and umbilical cord blood, which can produce IgM as a
first line of defense [29,31–34]. CD5+B cells are predominantly
found in the spleen and peripheral blood in chimeras . Little is
known about the phenotypes of human CD19+
B cells and
Transplant Immunology 26 (2012) 156–162
⁎ Corresponding author: Rui Sun, School of Life Sciences, University of Science and
Technology of China, 443 Huangshan Road, Hefei, Anhui 230027, China. Tel.: +86 551
360 7977, fax: +86 551 360 6783.
E-mail address: email@example.com (R. Sun).
0966-3274/$ – see front matter © 2011 Elsevier B.V. All rights reserved.
Contents lists available at SciVerse ScienceDirect
journal homepage: www.elsevier.com/locate/trim
CD5+CD19+B cells in different hematopoietic organs of human
UCB-chimeric mice that reflect the developmental course of human
B cells in human UCB-chimeric mice.
Here, we describe the developmental stages and functions of B
cells in NOD/SCID mice transplanted with human CD34+cells. The B
cells in the peripheral organs were more mature than those in the
bone marrow. Only IgM natural antibodies were produced by the
human B cells in the human UCB-chimeric mice, and CD5+B cells
age of IL-10-producing B cells after LPS stimulation, possibly implying a
regulatory function for this B cell subset.
2. Materials and methods
NOD/LtSzPrkdcscid/J (NOD/SCID) mice were purchased from SLAC
laboratory animal company in Shanghai, the Institute of Laboratory
Animal Science at the Chinese Academy of Medical Sciences (CAMS),
and Peking Union Medical College (PUMC) in Beijing. Breeding pairs
and experimental mice were housed in microisolator cages under spe-
cific pathogen-free conditions in the animal facility of the School of
Life of the University of Science and Technology of China. The mice
were maintained on an irradiated sterile diet and given autoclaved,
acidified water. All of the experiments were performed according to
the guidelines outlined in the Guide for the Care and Use of Laboratory
2.2. CD34+cell purification
Human umbilical cord blood was collected during normal full-
term deliveries from Anhui Shengli Hospital after obtaining informed
consent. Cord blood mononuclear cells (CBMCs) were separated by
Ficoll-Hypaque (Solarbio, Shanghai, China) density gradient centrifu-
gation. Human CD34+hematopoietic stem cells were enriched with
anti-human CD34 magnetic microbeads (Miltenyi Biotec, Bergisch
Gladbach, Germany) according to the manufacturer's instructions.
The cells were further stained with FITC-anti-hCD34 antibodies (BD
PharMingen) or with isotype-matched mAbs FITC-IgG1, κ isotype
(BD PharMingen). The purity was evaluated by flow cytometry and
was >90% in all of the experiments.
2.3. Transplantation of human CD34+cells into NOD/SCID mice
NOD/SCID mice were pretreated with 400 μL of phosphate-
buffered saline containing 30 μL of anti-asialo GM1 antibody (Wako,
Osaka, Japan) 24 h before cell transplantation. The mice were injected
with 1.0×105CD34+cells through the tail vein 4 to 6 h after 200 cGy
total body irradiation. After transplantation, the mice were treated
with anti-asialo GM1 antibody every seven days for two weeks.
2.4. Measurement of human immunoglobulin in the plasma of the
Human IgM and IgG concentrations in the plasma of the recipient
mice were measured by a standard enzyme-linked immunosorbent
assay (ELISA). The ELISA kits were purchased from the Senxiongbio-
tech Company in Shanghai. For detection of the HBsAg-specific
human antibodies, ten recipient mice were immunized with 10 ng
of HBsAg (Kangtai, Shenzhen) emulsified in aluminum hydroxide
and then boosted once after two weeks. Plasma from the
HBsAg-immunized mice was harvested 2 weeks after the second im-
munization. The HBsAg-specific antibody was measured by radioim-
munoassay (RIA) using a kit purchased from the Beijing North
Institute of Biological Technology in Beijing.
2.5. Flow cytometric analysis of transplanted NOD/SCID mice
At the specified times after transplantation, the peripheral blood,
spleens, and bone marrow were harvested from the human
UCB-chimeric mice. Mouse leukocytes were analyzed by FITC-mCD45
APC-hCD45 (BD PharMingen). Human B lymphoid cells were evaluated
by FITC-hCD19, PE-hCD10, PE-hCD20, Percp-Cy5.5-hCD20, APC-hCD5,
PE-hIgM, PE-hIgD, PE-hIgG, Percp-Cy5.5-hCD38, FITC-hCD24 and PE-
Cy7-hCD19 (BD PharMingen). The analyzed cells were also stained
with isotype-matched mAbs for the above-mentioned antibodies to ex-
clude non-specific staining.
2.6. Immunohistochemical staining
Splenic tissues of micewere fixed with 4% paraformaldehyde, dehy-
drated with graded alcohol, embedded in paraffin,and stained with he-
matoxylin and eosin. NOD/SCID mouse spleens were used as controls.
For the phenotypic analysis of the human lymphocytes in the spleens
of recipient mice, paraformaldehyde-fixed paraffin-embedded sections
were immunostained with mouse anti-hCD19, anti-hCD3, and anti-
hCD11c antibodies (BD PharMingen) after treatment with heated
citrate buffer for antigen retrieval. Staining was shown after incuba-
tion with biotinylated goat anti-mouse secondary antibody and HRP-
conjugated streptavidin (Zhongshan Goldbridge Biotechnology, Bei-
jing), followed by reaction with diaminobenzidine substrate. All of
the samples were counterstained with hematoxylin.
2.7. IL-10 production by B cells after stimulation with LPS
To measure human IL-10 in the culture supernatants, 1×106total
cells isolated from the bone marrow or spleen of the human
UCB-chimeric mice were stimulated with LPS (10 μg/mL, Escherichia
coli serotype 0111:B4, Sigma) in 0.2 ml of complete medium in a
96-well flat-bottom plate for 72 h. The culture supernatant fluid was
collected and assayed using a human IL-10 ELISA kit (Senxiongbiotech,
Shanghai). To detect the intracellular human IL-10 by flow cytometric
analysis, 1×106isolated cells were resuspended with LPS (10 μg/mL)
for 72 h; PMA (50 ng/mL; Sigma), ionomycin (1 μg/mL; Sigma), and
monensin (2 μM; Sigma) were added for the last 6 h. Cells were har-
vested and blocked with mouse antiserum before staining with
hCD24 (BD PharMingen) and then fixed and permeabilized with Fix-
ation/Permeabilization Diluent (eBioscience). The permeabilized
cells were stained with PE-hIL-10 mAb (BD PharMingen) or PE-Rat
IgG1 (BD PharMingen) as an isotype control.
2.8. Statistical analysis
All of the data are shown as the mean±standard error of the
mean (SEM). The significance of differences was determined with
the Student's t-test; the significance level was set at 0.05.
3.1. Human CD45+cells develop in NOD/SCID mice after transplantation of human UCB-
To ensure that the human CD34+cells can differentiate into human leukocytes in
NOD/SCID mice, high purity human CD34+cells from fresh umbilical cord blood
(Fig. 1A) were transplanted into NOD/SCID mice, and human CD45+cells were later
detected in different organs. The FACS analysis results showed a high percentage of
human CD45+lymphocytes (74.59%±8.97%) in the bone marrow after gating the lym-
phocyte region (Fig. 1B). Other hematopoietic organs, including the spleen
(28.24%±12.87%), peripheral blood (17.67%±9.36%) and liver (21.94%±10.56%),
also contained human CD45+cells (Fig. 1B); however, the proportions of CD45+
cells in the spleen and peripheral blood were lower than those in the BM in all of the
recipient mice (Fig. 1C). Hematoxylin and eosin staining showed that the spleens of
the human UCB-chimeric mice contained more cell clusters than did NOD/SCID mice
X. Wang et al. / Transplant Immunology 26 (2012) 156–162
(Fig. 1D), indicating the possible formation of primary lymphoid follicles. Taken to-
gether, these results indicate that human CD34+cells from human umbilical cord
blood can differentiate into human lymphocytes in immune-deficient mice.
3.2. CD19+cells predominate in human UCB-chimeric mice
To evaluate the ability of hematopoietic stem cells to differentiate into lympho-
cytes, we analyzed the subsets of lymphocytes from different organs of the recipient
mice. Few human CD3+T cells and CD56+NK cells were detected in the BM, spleen,
liver or peripheral blood; however, human CD11c+cells were detected in the bone mar-
row, spleen and liver of some of the human UCB-chimeric mice, although very few were
found in the peripheral blood. In contrast, human CD19+B cells were readily detected in
all of the organs analyzed (Fig. 2A). Immunohistochemical staining showed that there
were many B cells in the spleen but few dendritic cells and no T cells (Fig. 2C), which
was consistent with the results found by flow cytometry (Fig. 2B). Statistical analysis also
showed that the human CD19+cells were the predominant type of lymphocyte in the
Fig. 1. Human CD45+cells in NOD/SCID mice after transplantation of human UCB-CD34+cells.UCB-CD34+cells were isolated from human cord blood mononuclear cells (CBMCs).
NOD/SCID mice were pretreated with α-ASGM1 antibody to deplete the NK cells. After 24 h, the mice were irradiated and then received intravenous injections of 1.0×105human
bone marrow,spleen, and peripheral blood of NOD/SCIDmice at 16 weeks after transplantation.(C) Statistical analysis of human CD45+cells. Valuesare shown as the mean±SEM from
six mice at the 16 week time point. (D) Spleen histology was determined by H&E staining of the UCB-chimeras and untransplanted NOD/SCID mice (200×, final magnification). Yellow
arrows indicate the cell clusters in the spleen. The data shown are representative of three independent experiments.
0.4872.34 71.441.37 12.68 60.13
CD19 CD3 CD11c
BM SpleenCD45 CD19CD45 CD19
Fig. 2. CD19+cells are dominant in human UCB-chimeric mice.NK-depleted-NOD/SCID mice were irradiated and then received intravenous injections of 1.0×105human
UCB-CD34+cells. Human MNCs were analyzed 16 weeks after the transplantation of human UCB-CD34+cells. (A) MNCs were isolated from the bone marrow, spleens, peripheral
bloodand liversofthehuman UCB-chimericmice.The percentages ofCD19+cells,CD3+cells,CD11c+cells and CD56+cells inthehumanCD45+cells wereanalyzedbyflowcytometry.
(B) Splenic human B cells (CD19), T cells (CD3) and DCs (CD11c) were detected by flow cytometry and (C) by immunohistochemical staining (200×, final magnification) in the same
human UCB-chimeric mice. Yellow arrows indicate the human CD19+cells (left panel) and CD11c+cells (middle panel) in the spleen. (D) Statistical analysis of the percentage of
humanCD45+cells and CD19+cells inthe BMand spleen. Valuesare shownas themean±SEM from sixmice atthe 16 week time point. The ratioof CD19+/CD45+cells was calculated.
X. Wang et al. / Transplant Immunology 26 (2012) 156–162
bone marrow andspleen(Fig.2D). Together,these results indicate that human CD34+cells
can develop into certain types of lymphocytes in NOD/SCID mice, the majority of which are
3.3. Mature B cells accumulate in peripheral immune organs of human UCB-chimeric mice
Although the majority of the human CD45+cells were B cells, the matura-
tional state of B cells in human UCB-chimeric mice is not well known. Compared
with the B cells in the umbilical cord blood (Fig. 3A), a high percentage of the B
cells in the BM were CD10hi, IgM−, IgD−,and CD20−, and only a low propor-
tion were IgM+, IgD+,and CD20+, manifesting a phenotype of immature B cells
or even progenitors. In the spleen, nearly half of the B cells were mature B cell
phenotypes that were CD20+, IgM+and IgD+, and most of the B cells in the pe-
ripheral blood were highly mature (Fig. 3B). Together, these results indicate that
the development of Bcellsisdifferentinthebonemarrow,thespleenandtheperipheral
6.60 93.4051.46 48.54
28.10 71.90 13.73 86.27 99.82 0.180.58 98.73 87.5412.46
70.28 29.72 67.52 32.4899.86 0.14 0.31 99.69 14.55 85.45
93.806.2099.88 0.12 16.77 83.23 0.00 100.00
IgG IgDIgMCD20 CD10
Fig. 3. Mature B cells accumulate in peripheral immune organs of human UCB-chimeric mice.Human CD19+cells were gated from the MNCs of CBMCs or different organs of human
UCB-chimeric mice. CD19+cells were analyzed by flow cytometry for the expression of developmental markers on the cells, including IgM, IgD, IgG, CD10 and CD20. The data
shown are representative of three independent experiments. (A) MNCs were isolated from human umbilical cord blood. (B) NK-depleted-NOD/SCID mice were irradiated and
then received intravenous injections of 1.0×105human UCB-CD34+cells. MNCs were isolated from the bone marrow (upper), spleen (middle) and peripheral blood (lower) of
the human UCB-chimeric mice 16 weeks after injection with human UCB-CD34+cells.
IgD IgMCD20CD10 CD5
Fig. 4. Mature B cells are CD24intCD38intin human UCB-chimeric mice.Human CD19+cells were gated from the MNCs and then divided into two subpopulations, including
CD24hiCD38hiand CD24intCD38intcells. The expression of CD5 and developmental markers, including IgM, IgD, CD10 and CD20, were compared with human CD24hiCD38hiand
CD24intCD38intcells from CBMCs and different organs of the human UCB-chimeric mice. The data shown are representative of three independent experiments. (A) MNCs were isolated
from human umbilical cord blood. (B) NK-depleted-NOD/SCID mice were irradiated and then received intravenous injections of 1.0×105human UCB-CD34+cells. MNCs were isolated
from the bone marrow (upper), spleen (middle) and peripheral blood (lower) of human UCB-chimeras mice 16 weeks after transplantation of the human UCB-CD34+cells.
X. Wang et al. / Transplant Immunology 26 (2012) 156–162
3.4. Mature B cells are CD24intCD38intin human UCB-chimeric mice
We analyzed the CD19+CD38hiCD24hiB cells and CD19+CD38intCD24intB cells in the
human UCB-chimeric mice. In the bone marrow, most of the human CD19+cells were
CD24hiCD38hiCD10hibut IgM−IgD−CD20−; the CD24intCD38intCD19+cells were very
few in number. In the spleen, the proportion of CD19+CD38intCD24intB cells was
increased. The expression of IgM, IgD and CD20 on the CD19+CD38intCD24intB
cells was higher than on the CD19+CD38hiCD24hiB cells, but the expression of CD10 on
the CD19+CD38intCD24intB cells was lower. The phenotypes of the two subsets of
human B cells in the peripheral blood were similar to those found in the spleen
(Fig. 4B). In CBMC, most of the human B cells were CD19+CD38intCD24int, whereas a
few were CD19+CD38hiCD24hi(Fig. 4A). Therefore, CD38hiCD24hiimmature B cells
were principally found in the bone marrow, whereas CD38intCD24intmature B cells
were found in the spleen and peripheral blood.
3.5. IgM is the major antibody in the plasma of human UCB-chimeric mice
The spleen and peripheral blood contain many human mature B cells in human
UCB-chimeric mice, but their functionality is unknown. Thus, the level of human im-
munoglobulin in the plasma of unvaccinated human UCB-chimeric mice was mea-
sured. IgM was detected, but IgG was not detected (Fig. 5A), and the concentration
of IgM was positively related to the number of B cells (Fig. 5B). When the human
UCB-chimeric mice were immunized with HBsAg vaccine, only two in ten mice pro-
duced HBsAg-specific antibodies (Fig. 5C, D). The class of specific antibody in the two
human UCB-chimeric mice could not be determined due to the shortcomings of the
RIA kit. Together, these results show that most of the B cells in the spleens of the
human UCB-chimeric mice were IgM-producing B cells.
3.6. CD5+cells predominate among the mature CD19+cells of human UCB-chimeric mice
B1 cells are the main producers of IgM, thus CD5+CD19+B cells were detected in
the lymphocytes from different immune organs of the human UCB-chimeric mice. In
the bone marrow, only a small number of CD19+B cells were CD5 positive, but
many CD19+CD5+B cells were detected in the spleen and peripheral blood (Fig. 6A,
B). As shown in Fig. 6A and B, a large proportion of the CMBC B cells were CD5 positive.
The CD5+CD19+B cells in the investigated organs were CD24intCD38int(Fig. 4B) and
mature B cells (Fig. 6C). The phenotypes and distributions of
CD5+CD19+cells are shown in Table 1. Altogether, these results indicate that almost
all of the mature B cells in the human UCB-chimeric mice were CD5+B cells.
% B cells
Fig. 5. IgM is the major antibody in the plasma of the human UCB-chimeric
mice.NK-depleted-NOD/SCID mice were irradiated and then received intravenous
injections of 1.0×105human UCB-CD34+cells. (A) Plasma was harvested from the
PBL of the human UCB-chimeric mice 12 weeks after transplantation of the human
UCB-CD34+cells. The concentrations of human total IgM and IgG in the plasma were
between IgM concentration and the percentage of B cells from the same mouse was
analyzed. The data shown are from four mice. (C) Ten human UCB-chimeric mice
were immunized with 10 ng of HBsAg emulsified in aluminum hydroxide 12 weeks
nization. The percentage of mice (out of ten mice) exhibiting HBsAg-specific antibodies
was calculated. (D) The concentration of HBsAg-specific antibody in two mice was mea-
sured by RIA.
74.98 1.73 24.5420.2616.2311.23 6.41 2.23
BMSpleen Blood CBMCs
BMSpleen Blood CBMC
Fig. 6. CD5+cells are dominant in mature CD19+cells of human UCB-chimeric mice.MNCs were isolated from human umbilical cord blood or from the bone marrow, spleen and
peripheral blood of NK-depleted-NOD/SCID UCB-chimeric mice 16 weeks after transplantation. (A) The percentages of human CD5+CD19+cells in the CBMCs and UCB-chimeric
mouse MNCs were analyzed by flow cytometry. The data shown are representative of three independent experiments. (B) Statistical analysis of the percentages of human
CD5+CD19+cells in the CD19+cells from the CBMCs and UCB-chimeric mouse MNCs. Data are shown as the mean±SEM. (C) The expression of IgM, IgD, CD10 and CD20 on
human CD19+CD5+cells from the bone marrow (upper), spleen (middle) and peripheral blood (lower) were analyzed by flow cytometry in the human UCB-chimeric mice.
The data shown are representative of three independent experiments.
X. Wang et al. / Transplant Immunology 26 (2012) 156–162
3.7. Human B cells produce IL-10 after stimulation with LPS
To clarify whether the human B cells in the human UCB-chimeric mice had regula-
tory potential, cells isolated from the bone marrow and spleen were stimulated with
LPS. The results showed that the B cells from either the bone marrow or spleen pro-
duced human IL-10 (Fig. 7A). We also probed for intracellular human IL-10 in the
CD24+cells, which indicated that the human IL-10 was from the B cells (Fig. 7B).
After 72 h of stimulation, human IL-10 was measured in the culture supernatant. The
production of human IL-10 in the bone marrow group was much higher than that
from the spleen group, partly due to the greater number of B cells in the bone marrow
(Fig. 7C). TheseresultsindicatethattheBcellsinthehumanUCB-chimericmicemayhave
a regulatory function of producing IL-10.
The in vivo study of the development and roles of human B cells
has been hindered by the lack of proper model systems. In this
study, we investigated the development and function of human B
cells in umbilical cord blood cell-transplanted NOD/SCID mice. Begin-
ning 6 weeks after transplantation with CD34+cells, human CD45+
cells were detected in the peripheral blood, and the percentage of
human CD45+cells gradually increased. The cell clusters in the spleens
of the human UCB-chimeric mice were much more prevalent than in
the untransplanted NOD/SCID mice. These results demonstrated that
the microenvironment could influence the human CD34+cells to de-
velop into human CD45+leukocytes in the NOD/SCID mice. However,
human T cells were detected only rarely, if at all, in the NOD/SCID
mice transplanted with CD34+cells, which has also been reported in
previous studies [10,12,23]. The development of T cells includes both
a positive selection and a negative selection. It is possible that human
T cell progenitors may be unable to migrate into the mouse thymus in
vivo or to obtain sufficient nutrients and signals that are important for
T cell development in NOD/SCID mice.
B cells were the major component of the human CD45+cells in the
bone marrow, spleens and peripheral blood of the recipient mice, but
their developmental stages differed among the immune organs, which
is in accordance with other investigations [10,12,23]. Whereas most of
the B cells in the bone marrow were a CD19+CD10hiCD20−IgM−IgD−
immature population, CD19+CD20+IgM+IgD+mature B cells predo-
minated in the spleen and peripheral blood. Immature and mature B
cells can be sorted into their respective phenotypes based on CD24
and CD38. The B cells in the bone marrow were largely CD24hiCD38hi,
whereas relatively few were CD24intCD38int. The CD24intCD38intB
cells in the spleen and peripheral blood were more mature than the
CD24hiCD38hiB cells, as suggested by the expression of IgM, IgD and
CD20. These resultssuggestthatthe bonemarrow is the primarydevel-
opmentalsite of humanB cells in NOD/SCIDmice, and that the spleenis
thematurationalsite.Wefound that CD5+CD19+B cellswere themost
abundant population of human B cells in the spleen and peripheral
blood but were less prevalent in the bone marrow, which is consistent
portion of CD5+B cells is high during fetal life and childhood but low
during adult . Consistent with our results, the percentage of B cells
expressing CD5 is also high in human umbilical cord blood . In
mice, CD5+B cells are prevalent in the peritoneal cavity but very in-
frequent in the spleen and peripheral blood . As shown in Fig. 4B,
CD5+B cells in the spleen and peripheral blood were largely
CD24intCD38intmature B cells, and the phenotypes of the CD5+B
the CD5+B cells in thespleen andperipheral blood and alsoin thebone
cells were the predominant component in the peripheral organs of
Maturation, phenotypes and distributions of human B cells in human UCB-chimeric mice.
Fig. 7. Human B cells produce IL-10 after stimulation with LPS.NK-depleted-NOD/SCID mice were irradiated and then received intravenous injections of 1.0×105human UCB-CD34+cells.
MNCswereisolatedfromthebonemarrowandspleen16 weeksafterinjectionofhumanUCB-CD34+cells.TheMNCswereculturedwithLPSfor72 h,andIL-10productionwasdetectedby
intracellularstainingandELISA.(A)IntracellularstainingofhumanIL-10inMNCs.TheMNCswereculturedwithLPSfor72 h,andPMA,ionomycin,andmonensinwereaddedforthelast6 h
with LPS for 72 h, and PMA, ionomycin, and monensin were added for the last 6 h before permeabilization and staining for IL-10. The cells were gated on CD24+cells. The data shown are
representativeof three independent experiments. (C) The concentration of human IL-10 produced by thecells from the bone marrow and spleensof the chimeras. TheMNCs were cultured
with LPS for 72 h. Culture supernatants were assayed with a human IL-10 ELISA kit. Data are shown as the mean±SEM and are representative of three independent experiments.
X. Wang et al. / Transplant Immunology 26 (2012) 156–162
human UCB-chimeric mice have not been clearly explained. One possi- Download full-text
and differentiation [23,31]. It is also possible that the particular micro-
environment of the recipient mice, especially the absence of functional
suggest that both cord blood CD34+cells and the microenvironment of
NOD/SCID mice together cause a high percentage of CD5+B cells.
the serum of the human UCB-chimeric mice, although little IgG was
detected. Importantly, the concentration of IgM in the plasma was cor-
related with the proportion of human CD45+cells, human CD19+B
cells and CD5+B cells in the human UCB-chimeric mice, suggesting
that human IgM was produced by the CD5+B cells. Moreover, we
showed that antigen-specific immunoglobulins were not produced by
the human B cells in eight out of ten of the human UCB-chimeric mice
afterimmunization withHBsAg. The class of antigen-specific Ig produc-
tion in two of the mice was possibly IgM due to the lack of IgG produc-
tion in the absence of T cells. Recently, it was reported that B cells can
negatively regulate immune responses in inflammation models by se-
creting IL-10. In vitro experiments have shown that B cells can produce
IL-10 after stimulation with LPS , CpG [38–40], or CD40 mAb
[40,41]. In human UCB-chimeric mice, B cells from the bone marrow
and spleen express and secrete a large amount of IL-10 when treated
with LPS, and it seems that the CD24hiCD38hiB cells more strongly pro-
duced IL-10. Whether the B cells in human UCB-chimeric mice could
work as a regulatory player in vivo requires further investigation.
In summary, we have described a population of CD19+cells that is
highly represented in the hematopoietic organs of NOD/SCID mice
transplanted with human CB CD34+cells. There is a high content of
immature B cells in the bone marrow with an IgM−IgD−CD20−CD24-
hiCD38hiphenotype and a high content of mature B cells in the spleen
and peripheral blood with an IgM+IgD+CD20+CD24intCD38intpheno-
type. Most of the B cells in the spleen are CD5+B cells, possessing the
ability to secrete IgM but not IgG. After stimulation with LPS, the B cells
fore, the humanized NOD/SCID xenogeneic chimeras may provide a use-
ful tool to study the origin, development, and function of B cells in vivo.
The authors declare no conflict of interests. This work was sup-
ported by the Natural Science Foundation of China (#31021061)
and the Ministry of Science & Technology of China (973 Basic Science
Project # 2007CB815805).
 Legrand N, Weijer K, Spits H. Experimental models to study development and
function of the human immune system in vivo. J Immunol 2006;176:2053–8.
 Manz MG. Human-hemato-lymphoid-system mice: opportunities and challenges.
 Shultz LD, Ishikawa F, Greiner DL. Humanized mice in translational biomedical
research. Nat Rev Immunol 2007;7:118–30.
 Macchiarini F, Manz MG, Palucka AK, Shultz LD. Humanized mice: are we there
yet? J Exp Med 2005;202:1307–11.
 Payne KJ, Crooks GM. Immune-cell lineage commitment: translation from mice to
humans. Immunity 2007;26:674–7.
 McCune JM, Namikawa R, Kaneshima H, Shultz LD, Lieberman M, Weissman IL.
The SCID-hu mouse: murine model for the analysis of human hematolymphoid
differentiation and function. Science 1988;241:1632–9.
 Mosier DE, Gulizia RJ, Baird SM, Wilson DB. Transfer of a functional human im-
mune system to mice with severe combined immunodeficiency. Nature
 Lapidot T, PflumioF, Doedens M, MurdochB, Williams DE, Dick JE. Cytokine stimula-
tion of multilineage hematopoiesis from immature human cells engrafted in SCID
mice. Science 1992;255:1137–41.
 Pflumio F, Izac B, Katz A, Shultz LD, Vainchenker W, Coulombel L. Phenotype and
function of human hematopoietic cells engrafting immune-deficient CB17-severe
combined immunodeficiency mice and nonobese diabetic-severe combined im-
munodeficiency mice after transplantation of human cord blood mononuclear
cells. Blood 1996;88:3731–40.
 Hogan CJ, Shpall EJ, McNulty O, McNiece I, Dick JE, Shultz LD, et al. Engraftment
and development of human CD34(+)-enriched cells from umbilical cord blood
in NOD/LtSz-scid/scid mice. Blood 1997;90:85–96.
 Kollet O, Peled A, Byk T, Ben-Hur H, Greiner D, Shultz L, et al. beta2 microglobulin-
deficient (B2m(null)) NOD/SCID mice are excellent recipients for studying human
stem cell function. Blood 2000;95:3102–5.
 Ueda T, Yoshino H, Kobayashi K, Kawahata M, Ebihara Y, Ito M, et al. Hematopoietic
repopulating ability of cord blood CD34(+) cells in NOD/Shi-scid mice. Stem Cells
 Yoshino H, Ueda T, Kawahata M, Kobayashi K, Ebihara Y, Manabe A, et al. Natural
topoietic stem cell engraftment in NOD/Shi-scid mice. Bone Marrow Transplant
 Ito M, Hiramatsu H, Kobayashi K, Suzue K, Kawahata M, Hioki K, et al. NOD/SCID/
gamma(c)(null) mouse: an excellent recipient mouse model for engraftment of
human cells. Blood 2002;100:3175–82.
reconstitution of human lymphocytes from cord blood CD34+ cells using the
NOD/SCID/gammacnull mice model. Blood 2003;102:873–80.
 Traggiai E, Chicha L, Mazzucchelli L, Bronz L, Piffaretti JC, Lanzavecchia A, et al.
Development of a human adaptive immune system in cord blood cell-
transplanted mice. Science 2004;304:104–7.
 Ishikawa F, Yasukawa M, Lyons B, Yoshida S, Miyamoto T, Yoshimoto G, et al.
Development of functional human blood and immune systems in NOD/SCID/IL2
receptor gamma chain(null) mice. Blood 2005;106:1565–73.
 Shultz LD, Lyons BL, Burzenski LM, Gott B, Chen X, Chaleff S, et al. Human lymphoid
and myeloid cell development in NOD/LtSz-scid IL2R gamma null mice engrafted
with mobilized human hemopoietic stem cells. J Immunol 2005;174:6477–89.
 Lan P, Tonomura N, Shimizu A, Wang SM, Yang YG. Reconstitution of a functional
human immune system in immunodeficient mice through combined human fetal
thymus/liver and CD34(+) cell transplantation. Blood 2006;108:487–92.
of primitive human hematopoietic cells capable of repopulating NOD/SCID
mouse bone marrow: implications for gene therapy. Nat Med 1996;2:1329–37.
of the functions of human B and T cells in humanized NOD/shi-scid/gammac(null)
(NOG) mice (hu-HSC NOG mice). Int Immunol 2009;21:843–58.
 Noort WA, de Groot-Swings GMJS, Kester MGD, Melenhorst JJ, Willemze R,
Falkenburg JHF. Human (pre-) B-cells develop in bone marrow, but mature
in spleen of NOD/SCID mice after transplantation of CD34(+) cells from umbilical
cord blood (UCB). Blood 1998;92:116a-a.
generate CD5(+) B lymphoid cells in NOD/SCID mice. Stem Cells 1999;17:242–52.
 Shultz LD, Schweitzer PA, Christianson SW, Gott B, Schweitzer IB, Tennent B, et al.
Multiple defects in innate and adaptive immunologic function in NOD/LtSz-scid
mice. J Immunol 1995;154:180–91.
 Nagasawa T. Microenvironmental niches in the bone marrow required for B-cell
development. Nat Rev Immunol 2006;6:107–16.
entiation in the spleen of immunodeficient mice. J Immunol 2001;166:2929–36.
 Carsetti R, Rosado MM, Wardmann H. Peripheral development of B cells in mouse
and man. Immunol Rev 2004;197:179–91.
 Sims GP, Ettinger R, Shirota Y, Yarboro CH, Illei GG, Lipsky PE. Identification and
characterization of circulating human transitional B cells. Blood 2005;105:4390–8.
 Hayakawa K, Hardy RR. Development and function of B-1 cells. Curr Opin Immunol
 Hardy RR. B-1 B cell development. J Immunol 2006;177:2749–54.
 Kipps TJ. The CD5 B cell. Adv Immunol 1989;47:117–85.
 Bhat NM, Kantor AB, Bieber MM, Stall AM, Herzenberg LA, Teng NNH. The ontog-
eny and functional-characteristics of human B-1 (Cd5+B) cells. Int Immunol
 Kantor AB, Herzenberg LA. Origin of murine B cell lineages. Annu Rev Immunol
 Baumgarth N. The double life of a B-1 cell: self-reactivity selects for protective
effector functions. Nat Rev Immunol 2011;11:34–46.
 Nisitani S, Murakami M, Akamizu T, Okino T, Ohmori K, Mori T, et al. Preferential
localization of human CD5+ B cells in the peritoneal cavity. Scand J Immunol
 MatsumuraT,Kametani Y,AndoK,HiranoY, Katano I,Ito R,etal.Functional CD5+B
cells develop predominantly in the spleen of NOD/SCID/gammac(null) (NOG) mice
transplanted either with human umbilical cord blood, bone marrow, or mobilized
peripheral blood CD34+ cells. Exp Hematol 2003;31:789–97.
 Dalwadi H, Wei B, Schrage M, Spicher K, Su TT, Birnbaumer L, et al. B cell develop-
mental requirement for the G alpha i2 gene. J Immunol 2003;170:1707–15.
 Brummel R, Lenert P. Activation of marginal zone B cells from lupus mice with
type A(D) CpG-oligodeoxynucleotides. J Immunol 2005;174:2429–34.
 Lenert P, Brummel R, Field EH, Ashman RF. TLR-9 activation of marginal zone
B cells in lupus mice regulates immunity through increased IL-10 production.
J Clin Immunol 2005;25:29–40.
 Blair PA, Norena LY, Flores-Borja F, Rawlings DJ, Isenberg DA, Ehrenstein MR, et al.
but are functionally impaired in systemic Lupus Erythematosus patients. Immunity
 Zheng J, Liu YP, Lau YL, Tu WW. CD40-activated B cells are more potent than im-
mature dendritic cells to induce and expand CD4(+) regulatory T cells. Cell Mol
X. Wang et al. / Transplant Immunology 26 (2012) 156–162