Relatively Normal Human Lymphopoiesis but
Rapid Turnover of Newly Formed B Cells in
Transplanted Nonobese Diabetic/SCID Mice
This information is current as
of June 15, 2017.
Maria Isabel D. Rossi, Kay L. Medina, Karla Garrett, Grant
Kolar, Phillip C. Comp, Leonard D. Shultz, J. Donald Capra,
Patrick Wilson, Arthur Schipul and Paul W. Kincade
J Immunol 2001; 167:3033-3042; ;
doi: 10.4049/jimmunol.167.6.3033
http://www.jimmunol.org/content/167/6/3033
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References
Relatively Normal Human Lymphopoiesis but Rapid Turnover
of Newly Formed B Cells in Transplanted Nonobese Diabetic/
SCID Mice1
Maria Isabel D. Rossi,2* Kay L. Medina,2* Karla Garrett,* Grant Kolar,† Phillip C. Comp,‡
Leonard D. Shultz,§ J. Donald Capra,† Patrick Wilson,† Arthur Schipul,¶ and
Paul W. Kincade3*
M
any questions remain about molecular mechanisms responsible for formation and maintenance of the humoral immune system and it is unclear whether all
findings made in experimental animals can be extrapolated to humans. Gene targeting experiments and natural mutations suggest
that IL-7 is more important for B cell formation in mice than it is
in humans (1– 4). On the other hand, consequences of mutations in
the Btk tyrosine kinase gene are more severe for humans than for
mice (5, 6). Observations of that kind provide impetus for animal
models that would efficiently support formation of human B cells
from hemopoietic stem cells and nonobese diabetic (NOD)4/SCID
mice are extremely interesting in that context (7–9). Many laboratories have shown that B cells are formed in these animals from
transplanted human stem cells (8, 10 –12), but it remains unclear
how closely this engraftment reflects normal steady-state conditions. For example, it remains uncertain if a stable equilibrium is
attained where a pool of quiescent donor stem cells is established,
*Immunobiology and Cancer Program, †Molecular Immunogenetics, Oklahoma Medical Research Foundation, Oklahoma City, OK 73104; ‡Department of Medicine,
University of Oklahoma Health Sciences Center, Oklahoma City, OK 73104; §The
Jackson Laboratory, Bar Harbor, ME 04609; and ¶St. Anthony Hospital, Oklahoma
City, OK 73101
Received for publication April 6, 2001. Accepted for publication July 6, 2001.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
This work was supported by Grants AI20069, AI 33085, AI 45864, AI 30389, DK
53024 and DK, 57199 from the National Institutes of Health. P.W.K. holds the William H. and Rita Bell Chair in biomedical research.
2
M.I.D.R. and K.L.M. contributed equally to this work.
3
Address correspondence and reprint requests Dr. Paul W. Kincade, Immunobiology
& Cancer Program, Oklahoma Medical Research Foundation, 825 North East 13th
Street, Oklahoma City, OK 73104. E-mail address: [email protected]
4
Abbreviations used in this paper: NOD, nonobese diabetic; APC, allophycocyanin;
BrdU, 5-bromo-2⬘-deoxyuridine; EBF, early B cell factor; s, surface.
Copyright © 2001 by The American Association of Immunologists
if these stem cells continuously generate early lymphocyte precursors, and if the precursors progress through a normal sequence of
proliferation and differentiation steps. Alternatively, human B cells
might only be transiently produced as a wave of differentiation
from the engrafted precursors. Various checkpoints and quality
control events have been identified from animal studies, but we do
not know whether they pertain to this model. For example, human
lymphocytes might survive in chimeric mice without regard for the
presence and/or specificity of Ag receptors, whereas this is a rigorously controlled process under normal circumstances. Finally,
no information is available about the diversity of B cells in this
model and all could result from clonal expansion of a small number of precursors.
A thorough understanding and validation of the NOD/SCID chimera model should be invaluable for many purposes. A common
environment would be very useful for testing the differentiation
potential of stem cells from fetal, adult, and aged marrow sources.
Stem cells in some strains of mice undergo age-related changes
and this important issue begs study with respect to human stem
cells (13). Molecules within bone marrow and spleen serve as positive and negative regulators for the production, maintenance, and
function of B cells. Some that await identification might be effectively studied in the NOD/SCID model on the basis of species
specificity, by genetic manipulation, or by use of neutralizing Abs.
By exploitation of techniques developed for animal studies, new
insight should be obtained into the kinetics of production and turnover of human B cells.
Experimental animal studies suggest that B lymphopoiesis occurring during fetal life may differ in important ways from that
which occurs in adult marrow. A different repertoire of Ab variable
region genes is used and the B cells that express them differ from
adult B cells in other respects (14, 15). The more limited information available suggests this may also be the case for human B
cells (16), but we have little idea of its basis in either species.
0022-1767/01/$02.00
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Human B lineage lymphocyte precursors in chimeric nonobese diabetic/SCID mice transplanted with umbilical cord blood cells
were directly compared with those present in normal bone marrow. All precursor subsets were represented and in nearly normal
proportions. Cell cycle activity and population dynamics were investigated by staining for the Ki-67 nuclear Ag as well as by
incorporation experiments using 5-bromo-2ⴕ-deoxyuridine. Again, this revealed that human B lymphopoiesis in chimeras parallels
that in normal marrow with respect to replication and progression through the lineage. Moreover, sequencing of Ig gene rearrangement products showed that a diverse repertoire of VH genes was utilized by the newly formed lymphocytes but there was
no evidence for somatic hypermutation. The newly formed B cells frequently acquired the CD5 Ag and had a short life span in
the periphery. Thus, all molecular requirements for normal B lymphocyte formation are present in nonobese diabetic/SCID mice,
but additional factors are needed for recruitment of B cells into a fully mature, long-lived pool. The model can now be exploited
to learn about species restricted and conserved environmental cues for human B lymphocyte production. The Journal of Immunology, 2001, 167: 3033–3042.
3034
Again, it would be ideal to compare the fate of stem cells from
embryonic and adult sources in a common environment. B cells in
animals and humans are known to be heterogeneous in other ways.
Some of this diversity may result from functional specialization
and it has even been proposed that there are separate lineages of B
cell differentiation (17). Alternatively, heterogeneity of B cells
may result from encounters with Ag and participation in immune
responses (18 –20).
Our study was designed to learn whether the normal stages of
human B lymphopoiesis are represented in chimeric NOD/SCID
mice and that indeed appears to be the case. Precursor:product
ratios compared favorably to those present in fetal and adult marrow samples, suggesting a relatively normal progression through
this sequence. Furthermore, typical patterns of Ig gene rearrangements were documented. We conclude that the model provides
exceptional opportunities for investigation of human B cell formation. The short survival time of newly formed B cells raises questions about requirements for their maintenance in peripheral lymphoid tissues.
HUMAN B LYMPHOPOIESIS IN NOD/SCID MICE
BrdU administration
BrdU (Sigma) was administered to mice in the drinking water at a concentration of 1 mg/ml for different periods of time. This dose was based on
previous studies (21). The water bottle containing the BrdU solution was
protected from light and changed every 2–3 days throughout the time
course of the experiment. Alternatively, in short-term experiments, BrdU
was injected i.p. (1 mg/mouse) in PBS before the addition of BrdU to the
drinking water.
Intracellular and surface staining for FACS analysis
Cell sources
Cell sorting
Cord blood samples were obtained from placentas of healthy newborns
collected at Oklahoma University Hospital (Oklahoma City, OK). Adult
bone marrow samples were obtained from patients between 24 and 88
years of age and represented discarded material from hip replacement surgery at the Bone and Joint Hospital (Oklahoma City, OK). Fetal bone
marrow between 16 and 24 wk of gestation was supplied by the Anatomic
Gift Foundation (Laurel, MD). An Institutional Review Board approved all
studies involving human cells.
Bone marrow and spleen suspensions recovered from transplanted NOD/
SCID mice were incubated with anti-human CD5-PE and anti-human IgMAPC. Alternatively, bone marrow cell suspensions were stained with antihuman IgM-FITC, anti-human CD5-PE, and anti-human CD19-APC.
Single CD19⫹IgM⫺ or IgM⫹CD5⫹ or IgM⫹CD5⫺ cells were sorted into
each 96-well PCR plates using a MoFlo (Cytomation, Fort Collins, CO)
cell sorter with an automatic cell deposition unit. Bulk sorting of human
CD34⫹ hemopoietic cell subsets were conducted after depletion of
mouse lineage- positive cells from NOD/SCID bone marrow. Cells
were incubated with purified mAbs to Mac-1(M1/70), Gr-1(Ly-6G;
RB-8C5), and Ter119 (all purchased from BD PharMingen), followed
by incubation with goat anti-rat IgG-coated magnetic beads (Polysciences, Warrington, PA) followed by incubation with mAbs to human
CD34-FITC, CD10-PE, and CD19-APC. CD34 ⫹ CD10 ⫺ CD19 ⫺ ,
CD34⫹CD10⫹CD19⫺, and CD34⫹CD10⫹CD19⫹ cells were sorted
using a MoFlo. Postsort analyses revealed the purity of the sort
populations to be ⬎95%.
Materials and Methods
Animals
Transplantation of human cells
Cord blood samples were diluted 1:4 in HBSS (without Ca2⫹ and Mg2⫹)
before separation over Ficoll-Hypaque (Lymphocyte Separation Medium;
Cellgro, Herndon, VA). The mononuclear cell fraction from cord blood
was diluted in PBS with 0.1% BSA (BSA fraction V; Sigma, St. Louis,
MO) and 20 ⫻ 106 cells were injected into the tail vein of sublethally
irradiated (100 cGy from a 137Cs source) 8- to 12-wk-old mice.
Treatment with anti-IL 7
RT-PCR analyses of lymphoid genes
The M25 Ab that neutralizes murine and human IL-7 was generously provided by Dr. F. Finkelman (University of Cincinnati Medical Center, Cincinnati, OH). NOD/SCID mice injected previously with cord blood mononuclear cells were treated three times a week for 2 wk with 3 mg of this Ab.
Control mice received PBS. Three days after the last injection, the animals
were sacrificed and bone marrow cells were harvested for flow cytometry.
Sorted cells were put in TRIzol reagent (Life Technologies) and total RNA
was extracted following the manufacturer’s instructions. Alternatively, the
cells were put in lysis buffer (Ambion, Austin, TX) and mRNA was extracted using poly(A) columns (Ambion) according to the manufacturer’s
instructions. Total RNA or mRNA was treated with DNase I to remove
contaminating genomic DNA using the manufacturer’s instructions and
cDNA was prepared using oligo(dT) and Moloney murine leukemia virus
reverse transcriptase (Life Technologies) following standard protocols.
Semiquantitative PCR was done to measure relative differences in transcript levels of target cDNAs against levels of the reference gene GAPDH.
The PCR was done in 100 ␮l containing 1⫻ PCR buffer (Takara Biomedical, Osaka, Japan), 1.5 mM MgCl2, 200 ␮M dATP, dGTP, and dTTP, 100
␮M dCTP, and 50 pmol of each primer. Samples were overlaid with mineral oil. For quantification, 0.5 ␮Ci of [␣-32P]dCTP (Amersham, Arlington
Heights, IL) was included in each reaction tube. Samples were denatured
in a DNA thermocycler (PerkinElmer, Norwalk, CT) for 10 min at 95°C.
To increase specificity, 2.5 U of Taq DNA polymerase (Takara Biomedical) was added to each sample during this initial denaturation. Samples
were then cycled for 1 min at 94°C, 1-min annealing at 60°C, and 1.5-min
extension at 72°C with a final extension of 7 min at 72°C. Aliquots were
removed at cycles 25, 28, 31, and 34 for GAPDH and cycles 32, 35, and
38 for all others to ensure that PCR remained within the exponential range
of amplification. Aliquots (5 ␮l) were denatured in a formamide loading
Conjugated Abs for flow cytometry
The following Abs were either biotinylated or conjugated with FITC, PE,
or allophycocyanin (APC): anti-CD5 (clone UCHT2), anti-CD38 (clone
HIT2), anti-CD24 (clone ML5), anti-CD23 (clone M-L233), anti-CD43
(clone 1G10), anti-CD45 (clone HI30), anti-Ki-67 (clone B56), and antiIgD (clone IA6-2) from BD PharMingen (San Jose, CA); anti-CD34
(HPCA-2) and anti-5-bromo-2⬘-deoxyuridine (BrdU; clone B44) from BD
Biosciences (Mountain View, CA); anti-CD10 (clone 5–1B4), anti-CD19
(clone SJ25-C1), anti-CD13 (clone TUK1), anti-CD33 (clone 4D3), anti-␬
light chain (clone HP6062), anti-␭ light chain (clone HP6054), and antiglycophorin A (clone CLB-ery1) from Caltag Laboratories (Burlingame,
CA). F(ab⬘)2 of goat anti-human IgM or anti-human IgD were obtained
from Southern Biotechnology Associates (Birmingham, AL). The biotinylated Abs were revealed with Streptavidin Red 613 (Life Technologies,
Rockville, MD). TdT was detected using the FITC kit from Supertechs
(Bethesda, MD).
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NOD/LtSz-scid/scid (NOD/SCID) mice were obtained from a breeding
colony established at the Laboratory Animal Facility at the Oklahoma
Medical Research Foundation (Oklahoma City, OK) from breeding pairs
kindly provided by Dr. L. D. Schultz (The Jackson Laboratory, Bar Harbor,
ME). Animals were housed in a restricted barrier facility maintained in
microisolator cages under specific pathogen-free conditions.
Four-color immunofluorescence analysis was used for the identification of
the different B cell precursor populations. Single-cell suspensions from
bone marrow, spleen, and the peritoneal cavity were preincubated for 10
min on ice with unconjugated mouse Ig or mouse serum. The cells were
incubated for 15–30 min on ice with directly conjugated or biotinylated
Abs specific for human cell surface Ags, washed twice with PBS containing 3% FBS and 0.01% sodium azide (staining buffer), and incubated for
15 min on ice with the second-step reagent. Streptavidin Red 613 was used
to reveal the biotinylated Abs. The cells were then prepared for intracellular staining by fixation with 1% paraformaldehyde in PBS and permeabilized with 70% ethanol at ⫺20°C for ⬎30 min and washed twice with
the staining buffer. The cells were incubated with anti-human IgM Ab for
30 min at room temperature and TdT staining was done according to the
manufacturer’s instructions. To establish the proliferative fraction, cells
were incubated with the Ab anti-Ki-67 (mib-1) for 1 h on ice, as described
by Zupo et al. (22). To reveal BrdU, after fixation and permeabilization, the
cells were incubated in 1 ml of 0.15 M NaCl saline containing 4.2 mM
MgCl2, 10 ␮M HCl, and 100 U of DNase (Sigma) for 30 min at 25°C. The
cells were washed twice with buffer and stained with anti-BrdU as described elsewhere (23). Parallel staining and FACS analysis were done on
nontransplanted NOD/SCID mice to confirm that none of the human-specific Abs cross-reacted with murine cells.
The Journal of Immunology
buffer and applied to a 6% polyacrylamide gel containing 7 M urea. Incorporation of [␣-32P]dCTP into PCR product bands was quantified from
dry gels using a PhosphorImager (Molecular Dynamics, Sunnyvale, CA).
Primers were as follows: GAPDH sense: 5⬘-TCCAAAATCAAGT
GGGGCGAT-3⬘; GAPDH antisense: 5⬘-TTCTAGACGGCAGGTCAGGTC3⬘; 475-bp expected product, recombinase-activating gene (RAG) sense: 5⬘CCTGAGTCCTCTCATTGCTGAGAG-3⬘; RAG1 antisense: 5⬘-AGGGCA
TGATGATCGCCATACT-3⬘; 681-bp product, RAG2 sense: 5⬘-CTAATGA
AGAGCAGACAACATTCA-3⬘; RAG2 antisense: 5⬘-TAGGACTCT
TTGGGGAGTGTGTAG-3⬘; 422-bp product, Pax5 sense: 5⬘-CTCGGTG
AGCACGGATTCGGCC-3⬘; PAX-5 antisense: 5⬘-GCGGCAGCGCTATA
ATAGTAG-3⬘; 621-bp product, early B cell factor (EBF) sense: 5⬘-CCGGGC
TCACTTTGAGAAGCAG-3⬘; EBF antisense: 5⬘-CAGGGAGTAGCAT
GTTCCAGAT-3⬘, 638-bp product.
Single-cell PCR for VH gene analysis
Cloning and sequencing of Ig VH4 genes
More than 2000 cells were collected for each lymphocyte population and
mRNA was isolated using an oligo(dT) system (Ambion). Reverse transcription was conducted to produce cDNAs (Roche). PCR was then performed with the same VH4 external primer used for the single-cell PCR
experiments detailed above and a ␮ H chain constant region-specific
primer (5⬘-CTGGACTTTGCACACG-3⬘). Reaction conditions were the
same as those described above for the first gene-specific, single-cell analyses except that total cycles were reduced to 25 rounds of amplification.
PCR products were agarose gel purified (Qiagen, Valencia, CA) and transformed into Escherichia coli using a PCR-Blunt cloning kit (Invitrogen,
San Diego, CA). Successful transformations were identified by blue-white
screening and picked for overnight cultures from which plasmid DNA was
prepared (QIAprep Spin Miniprep kit; Qiagen) and sequenced on an Applied Biosystems 377 fluorescence automated DNA sequencer (Applied
Biosystems, Foster City, CA). Sequences were identified using the
DNAPLOT search components of the VBASE (http://www.mrc-cpe.cam.
ac.uk/imt-doc/, coordinated by I. M. Tomlinson, Medical Research Council
Centre for Protein Engineering Cambridge, U.K.) and International Immunogenetics Database (http://imgt.cines.fr, coordinated by M.-P. Lefranc,
Montpellier, France) databases. Further analysis was performed using the
Clustal X computer program for sequence alignments. Somatic mutation
was distinguished from Taq polymerase error by considering more than
two base pair changes per 300 nts to be somatic mutation. This cutoff was
calculated from the sequences of the constant regions obtained in these
studies. CDR3 length was defined as the interval between the consensus
cysteine residue of the V gene and the consensus tryptophan residue of the
J segment.
Results
Engraftment and normal differentiation of human cells in NOD/
SCID bone marrow
It has been previously shown that engraftment of SCID and NOD/
SCID mice requires conditioning treatment (25, 26). Our prelim-
inary experiments revealed that survival and human cell chimerism
were optimal with a low dose (100 cGy) of 137Cs irradiation, and
we confirmed that the majority of cells belonged to the B lymphocyte lineage (8, 11, 12, 27). Human CD45⫹ cells represented
39.4 ⫾ 17.6% of total bone marrow cells in these animals and
76.7 ⫾ 7.1% of them expressed CD19. In contrast, we found that
an average of 5.9 ⫾ 3.9% of the CD45⫹ cells in normal adult
marrow specimens was CD19⫹. Preferential development of B
lineage cells was also apparent at the CD34⫹ stage where 57.7 ⫾
7.0% of the human population expressed CD19. Additional analysis revealed that 79.8 ⫾ 6.9% were TdT⫹, 12.2 ⫾ 6.9% were
TdT⫹CD10⫺CD19⫺, and 6.6 ⫾ 2.4% were TdT⫹CD10⫹CD19⫺.
The importance of IL-7 to the development of human lymphocytes
was assessed by injection of the M25 monoclonal Ab capable of
neutralizing both murine and human cytokines. Human CD45⫹
cells represented 41.8 ⫾ 13.5% of total bone marrow cells in
treated animals and 76.3 ⫾ 7% of them were also CD19⫹.
Human B lymphopoiesis has been described in terms of sequential gain and loss of differentiation markers, and we used flow
cytometry to resolve various subsets in the chimeric bone marrow.
Expression of TdT is an early lymphoid lineage milestone and TdT⫹
cells were readily detected among the early CD34⫹CD10⫺CD19⫺
fraction (Fig. 1, A and B). CD34⫹CD10⫹CD19⫺ cells that are
potential common lymphoid progenitors (28) represented another
easily resolved category (Fig. 1, A and B), and RT-PCR analysis of
sorted marrow cells (Fig. 1, E–G) showed that four lymphocyteassociated genes were markedly up-regulated at that stage (Fig.
1D). As with their counterparts in normal human marrow (Refs. 29
and 30 and data not shown), the CD34⫹TdT⫹CD19⫹ pro-B cells
uniformly displayed CD10 (Fig. 1B).
Pre-B cells in chimeric bone marrow were resolved as the
cytoplasmic ␮ H chain⫹ surface ␬ or ␭⫺ fraction of CD19⫹
lymphocytes and further subdivided according to size (Fig. 2).
It can also be seen that most, but not all of the pre-B cells had
down-regulated CD34. The final steps in B lymphopoiesis are
marked by acquisition of Ig L chains, followed by surface (s)
IgD at the immature and naive B cell stages. Although both of
these human lymphocyte subsets were present in chimeric mice,
sIgM⫹sIgD⫹CD24lowCD38⫺CD10⫺ memory B cells that recirculate through normal adult marrow (31) were conspicuously absent (Fig. 3A). The B cells in chimeric bones closely resembled
those normally present in fetal marrow with respect to CD24 density along with display of CD38, CD10, and CD43 Ags (Fig. 3A
and data not shown). In addition, they were uniformly small (data
not shown). Small pre-B cells were overrepresented among the
CD19⫹ fraction of donor cells as compared with those normally
found in fetal and adult human bone marrow (Fig. 3B). However,
this difference was not significantly different. Otherwise, ratios between B lineage lymphoid compartments were remarkably like
those in normal human marrow. This represents the first direct
comparison of B lymphopoiesis in the animal model to normal
fetal and adult human marrow. We conclude that human hemopoietic cells preferentially expand and differentiate along the B
lymphocyte lineage in NOD/SCID mice. Although small pre-B
cells are elevated and memory B cells are absent, the entire series
of human B lineage differentiation is otherwise represented in
near-normal proportions.
Acquisition of CD5 by human B cells in chimeric animals
As expected from the marrow analysis, chimeric mice lacked a full
spectrum of mature B lymphocyte subsets in peripheral tissues. For
example, there were no CD38⫺ B cells in the spleen and most of
the cells displayed the high density of CD24 typical of newly
formed lymphocytes. As noted previously (12), many human B
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Single cells were directly deposited into 5 ␮l of an alkaline lysing solution
(200 mM KOH/50 mM DTT) in 96-well plates and subsequently neutralized with 5 ␮l of neutralization solution (900 mM Tris-HCl (pH 9.0), 300
mM KCl, and 200 mM HCl) (24). Genomic amplification was performed
using 2 ␮g of a random 15-mer primer, 400 ␮M dNTP Mix (Roche, Basel.
Switzerland), 5 U of Taq DNA polymerase (Promega, Madison, WI), 1.5
mM MgCl2, Taq polymerase buffer (final concentration 50 mM KCl, 10
mM Tris-HCl (pH 9.0), and 0.1% Triton X-100; Promega) in a final volume of 100 ␮l. This mixture was subjected to the cycling conditions described in Ref. 24. Five microliters of this material was then added to an
initial gene-specific reaction mix containing 100 ␮M dNTP mix, 5 U of
Taq DNA polymerase, 1.5 mM MgCl2, Taq polymerase buffer (final concentration 50 mM KCl, 10 mM Tris-HCl (pH 9.0), and 0.1% Triton X-100)
in a final volume of 50 ␮l. This mixture was then transferred to reaction
tubes (MBP BioProducts, San Diego, CA) containing 500 ng of each external primer (24) sealed in wax. Cycling conditions included an initial
4-min incubation at 95°C followed by 1 cycle of 1 min at 94°C, 1 min at
the lowest calculated primer annealing temperature, and 1 min at 72°C.
Thirty-nine subsequent cycles were performed where incubation at the
primer annealing temperature was decreased by 30 s and a final extension
incubation was performed for 10 min at 72°C. A second gene-specific PCR
using 10 ␮l of the product of the first gene-specific PCR was performed
using the same reaction mix added to an internal set of PCR primers (24)
sealed in wax. Cycling conditions were performed as described above for
the first gene-specific PCR amplification.
3035
3036
HUMAN B LYMPHOPOIESIS IN NOD/SCID MICE
FIGURE 1. Human B lineage lymphocytes
predominate in chimeric NOD/SCID mice. Mice
were prepared with low-dose irradiation before
transplantation with 20 ⫻ 106 human umbilical
cord blood mononuclear cells. Marrow cells
were recovered 7–10 wk later and analyzed by
flow cytometry. A, Most human CD34 cells express TdT. Numbers inside the quadrants represent percentages of CD34⫹ cells that are TdT⫹. B,
Human CD34⫹TdT⫹ cells were further characterized with respect to CD19 and CD10. C, Isotype
controls corresponding to CD10 and CD19 staining are also shown. D, RT-PCR was used to
evaluate B lineage-associated transcript levels in
human CD34⫹CD10⫺CD19⫺, CD34⫹CD10⫹
CD19⫺, and CD34⫹CD10⫹CD19⫹ cells. The
postsort analysis of these subsets of CD34⫹ cells
is shown in E–G.
FIGURE 2. Characteristics of human CD19⫹ cells recovered from
NOD/SCID bone marrow. Top left panel, Gates used for identification of
human CD19⫹ cells by flow cytometry. CD19⫹CD34⫹ pro-B and pre-B
cells are resolved with respect to cytoplasmic ␮ (c␮) and CD34 expression
in the top right panel. Lower left panel, Gates used to distinguish pre-B and
B cells, while the lower right panel demonstrates the large and small pre-B
cells resolved by light scatter. FSC, Forward scatter; SSC, side scatter.
SCID mice and they resemble normal neonatal B cells in some, but
not all respects.
Proliferation of human lympho-hemopoietic cells in chimeric
marrow
Until now, the extensive replication required for blood cell formation could only be satisfactorily investigated in experimental animals. Moreover, it was difficult to fully appreciate population dynamics in individual samples of human bone marrow. Proliferation
and turnover of lymphocyte populations have been extensively
studied in mice and rats following BrdU administration (32–35).
Therefore, human pre-B cells in chimeric marrow were discriminated by the presence of cytoplasmic ␮ H chains and absence of
surface Ig L chains before simultaneous analysis of BrdU incorporation and presence of the Ki-67 Ag (Fig. 5A). There was remarkably close agreement between these two indices of proliferation. That is, all of the large pre-B and 61% of the small pre-B
cells were mitotically active. The agreement between the two
methods made it possible to directly compare hemopoietic cell
expansion in transplanted animals to that in freshly obtained normal specimens (Fig. 5B). Proliferation of pro-B and pre-B cell
compartments most closely resembled that in adult human marrow.
The experiments were then expanded to include all stages of
lymphopoiesis. Hemopoietic stem cell activity in normal human
bone marrow is known to be associated with cells that are
CD34⫹CD38⫺/low (36). Most of these cells in human fetal and
adult bone marrow as well as those in cord blood are quiescent (36,
37). As others have found (8, 10), very small numbers (0.5 ⫾
0.5%) of human CD34⫹ cells in transplanted mice are CD38⫺/low
(Fig. 6A, left panel). In striking contrast to the situation in adult
and even fetal human bone marrow, most of these cells in chimeric
mice appeared to be actively proliferating when examined 7–10
wk after transplantation (Fig. 6A, middle panel). We separately
analyzed CD34⫹ cells that totally lacked CD38 and ones that had
a very low density of this marker (Fig. 6A, middle and right panels). Approximately 70% in each of these subsets expressed the
Ki-67 nuclear Ag associated with proliferating cells. Similar results were obtained when animals were observed 20 wk after transplantation. Since most of the human CD34⫹ cells in NOD/SCID
mice expressed CD19 and these pro-B cells are rapidly cycling
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cells in NOD/SCID mice express the CD5 Ag. We found that B
cells in chimeric bones closely resembled those in human fetal
marrow in this respect and that 70.8 ⫾ 10.5% of the B cells in
spleen were CD5⫹ (Fig. 4). CD5⫹ B cells were also recovered
from the peritoneal cavity of the mice, and particularly when the
extent of chimerism was high (data not shown). CD5⫹ B cells are
conspicuous in normal umbilical cord blood and we compared
them to B cells that arose in transplanted mice (Fig. 4, E–H).
Although CD5⫹ B cells from both sources were CD23⫺ and
CD11b⫺ (data not shown), those recovered from mice expressed
more CD10 and CD43 Ags, and densities of sIgD were lower.
Thus, impressive numbers of human B cells are produced in NOD/
The Journal of Immunology
3037
(Figs. 5B and 7A), BrdU incorporation was analyzed in CD34⫹
cells that lack CD19. Virtually all CD34⫹CD19⫺ cells were labeled after 1 wk of continuous administration (Fig. 6B). These
findings demonstrate that while typical patterns of lymphocyte precursor proliferation are seen in transplanted NOD/SCID mice, a
pool of quiescent CD34⫹CD38⫺ cells normally found in human
bone marrow is not established.
Turnover of human lymphoid cells in NOD/SCID mice
We then administered BrdU to NOD/SCID mice with transplants
to study population dynamics for human lymphoid cells. As might
be expected from the Ki-67 staining described above, large pre-B
cells had the fastest turnover and half were labeled in ⬍3 h (Fig.
7A). This would be compatible with a very high mitotic index and
short cell cycle time. Large pre-B cells are thought to derive from
the cycling pro-B cell compartment and we found that pro-B cells
were also rapidly labeled. We determined a 50% renewal time of
54 h for small pre-B cells and 105 h for sIgM⫹ B cells. Thus, there
was an interval of ⬎18 h between the last proliferating compartment and newly formed B cells. Although culture studies and patterns of tumor marker expression have previously suggested a
probable sequence of differentiation, these findings represent the
FIGURE 4. Acquisition of CD5 by a majority of
human B cells arising in NOD/SCID mice and comparison to cord blood lymphocytes. A–D, Display of
sIgM and the CD5 Ag is shown for cells falling within
the lymphocyte light scatter gate 9 wk after transplantation along with cells freshly harvested from normal
fetal marrow or cord blood. E–H, IgM⫹CD5⫹ B cells
recovered from chimeric spleen (filled histograms) are
compared with those from cord blood (open histograms). The dashed lines represent negative control
staining. These results are representative of those obtained in five independent experiments.
first kinetic analysis of human B lymphopoiesis in a marrow
environment.
These findings are compatible with the results of animal studies
in that the acquisition of sIg occurs without further cell division as
small pre-B become B cells within bone marrow (38). Only 3.6 ⫾
1.9% of human B cells in the chimeric spleens expressed even low
levels of Ki-67, suggesting there was little if any replication in that
site. However, we found progressively increasing BrdU incorporation for cells recovered from the spleen (Fig. 7B). Indeed, the
data were best described with a straight line (r2 ⫽ 0.97), indicating
that peripheral cells were homogeneous and relatively short-lived.
The calculated 50% renewal time for these B cells was between 7
and 8 days. Thus, although NOD/SCID marrow supports extensive
production of human B cells, the newly formed lymphocytes fail to
enter a long-lived pool.
Ig VH gene utilization in chimeric mice
All of the above findings suggest that human B lymphocytes are
actively produced in chimeric NOD/SCID mice, but do not exclude the possibility of oligoclonal expansion. Furthermore, our
understanding of human CD5⫹ B cells is incomplete. Therefore,
we used molecular techniques to investigate the status of the Ig VH
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FIGURE 3. Absence of mature, recirculating memory B cells in bone marrow of chimeric NOD/SCID mice and comparison to normal human specimens.
A, Recirculating memory cells are resolved on the basis of low densities of CD24, absence of CD10 and CD38, as well as the presence of sIgD. B, The
relative abundance of B lineage subsets in chimeras is compared with normal fetal and adult marrow. Gating used to make these determinations is shown
in Fig. 2. The immature, IgM⫹CD24highIgD⫺, and naive IgM⫹CD24highIgD⫹ B cells are included in one category. Data represent the mean results from
the analysis of 20 animals ⫾ SD.
3038
HUMAN B LYMPHOPOIESIS IN NOD/SCID MICE
Discussion
rearrangements in these lymphocytes. Single-cell PCR was conducted as an initial screen to determine whether a normal fraction
of Ig gene rearrangements utilized the VH4 family. The 34% frequency we obtained is typical for B cells in normal umbilical cord
blood (39). Therefore, we focused our analysis on the distribution
of rearrangements involving this well-characterized VH family.
Using a single set of primers, Ig transcripts were cloned and sequenced from pooled bone marrow and spleen cells (see Materials
and Methods). A total of 247 separate and distinct VH fragments
were sequenced, easily identified, and assigned to one of the
known VH4 genes or to a less frequently used gene, VH4-30-4. As
shown in Fig. 8, a normally diverse population of B lymphocytes
was produced in these chimeric animals. Indeed, the range of 27–
33% observed for VH4-34 is consistent with previous analyses of
freshly isolated cord blood B cells (39). Utilization of three specific gene segments, VH4-34, VH4-28, and VH4-30-4 was remarkably constant across the five lymphocyte populations analyzed.
Use of VH4-39 decreased with the exit of B cells from the marrow.
There was an interesting trend for increased utilization of VH4-59
and, to a lesser extent, VH4-04 with CD5 acquisition. A reciprocal
pattern, i.e., reduced usage by CD5⫹ B cells, was found for VH461. As might be expected from the absence of T lymphocytes in
these animals, we found no evidence for somatic hypermutation in
these VH genes.
These rearrangement products were further analyzed with respect to JH gene segment utilization and HCDR3 lengths. The JH4
segment is most commonly used by normal fetal (40) and cord
blood B cells (41), as was the case for 58% of the lymphocytes
analyzed from transplanted mice. The average of the mean
HCDR3 lengths for the population was 15.2 ⫾ 0.7 codons and falls
within limits previously described for cord blood and term infants
(42, 43). Each junction represented a unique sequence and there
was clear evidence for N nucleotide insertion, i.e., TdT activity.
Thus, these data show that NOD/SCID bone marrow supports all
aspects of human Ig gene rearrangement, generating a normally
diverse population of B cells.
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FIGURE 5. Mitotic activity of human lymphocyte precursors in grafted
mice closely resembles that in normal adult marrow and Ki-67 staining
provides a valid assessment. Cells expressing human CD34 and CD19 Ags
were gated in bone marrow (BM) harvested 7–10 wk after transplantation
and resolved into pro-B and pre-B cells as shown in Fig. 2. A, Flow cytometry of BrdU⫹ pre-B cells after 3 days of BrdU treatment is shown in
comparison to Ki-67 staining to evaluate the latter as an index of proliferation. B, Ki-67⫹ cells in chimeras are compared with those in freshly
isolated normal marrow specimens. Data represent the mean results from
the analysis of 18 animals ⫾ SD. FSC, Forward scatter.
The NOD/SCID model is now being extensively used as a functional assay for human stem cells. However, it also has great potential for understanding the normal steps, environmental requirements, and kinetics of B lymphopoiesis. The aim of this initial
study was to determine how closely the model reflects the events
that occur within normal fetal and adult bone marrow. We will
conclude that the entire differentiation series from stem cells to
immature B cells is represented. Relative numbers of cells at each
stage and proliferative expansion compare favorably to that within
normal human marrow. Moreover, a diverse population of Ag receptor genes is utilized. However, the newly formed B cells do not
appear to enter the long-lived pool.
Cord blood cells are becoming widely used and are uniquely
effective for clinical transplantation (44). Although it has been
shown that a quiescent CD34⫹CD38⫺ subset of umbilical cord
blood actually engrafts NOD/SCID mice, undefined accessory
cells improve the efficiency (7, 8, 10, 45). In preliminary experiments, we confirmed that was also the case with our transplantation protocol and observed no chimerism in mice injected with the
CD34⫺ fraction of cord blood cells. Stem cells from this source are
much more effective in this experimental model than ones harvested from either fetal or adult marrow (Refs. 26, 46, and 47 and
our unpublished observations). For all of these reasons, the entire
mononuclear fraction of cord blood was used in this initial study of
human B lymphopoiesis.
As others have found with this model (8, 11, 12, 27), human
hemopoietic cells preferentially expand within the B lymphocyte
lineage. However, this is the first detailed investigation of all
stages of B lymphopoiesis with a direct comparison to the replication and differentiation events that occur within normal marrow.
Stem cells with long-term repopulating potential are thought to be
nonreplicating and part of the CD34⫹CD38⫺ fractions of human
marrow or cord blood (7, 10, 48). Furthermore, this nonproliferating fraction contains the SCID mouse repopulating cells (49, 50).
Although donor type cells with this phenotype were readily identified in murine bone marrow, they appeared not to be quiescent
(Fig. 6). The time required for transplanted stem cells to exit the
cell cycle in a human marrow environment is not known and it is
therefore unclear whether our findings reflect species differences in
control over stem cell replication. However, the inability to establish a quiescent stem cell pool could account for the finding that
chimerism eventually declines in transplanted NOD/SCID mice
and an exhaustion of human stem cell activity is thought to occur
in these animals (26, 50, 51). TGF-␤, LIF, and macrophage-inflammatory protein 1␣ are among factors found to induce and/or
promote hemopoietic cell quiescence (52, 53). Since TGF-␤ is
conserved between mice and humans, some other species-specific
factor may be limiting in the recipient animals. Although not
within the scope of our study, the NOD/SCID model might be
manipulated to learn more about mechanisms that control stem cell
quiescence and self-renewal.
Commitment to the B lymphocyte pathway is a gradual process
and early precursors may express genes associated with other
blood cell lineages (54, 55). TdT is an extremely useful marker for
lymphocyte precursors in mice and can be detected in human marrow at a very early stage (30, 56). Here, we show that human
TdT⫹CD34⫹CD10⫺CD19⫺ cells represent a stable population in
chimeric bone marrow. Marked up-regulation of four genes required for B lymphocyte formation occurred at the presumptive
next stage of differentiation and this CD34⫹CD10⫹CD19⫺ fraction is thought to include the human counterparts of common lymphoid progenitors (28). That is, cells with these characteristics may
The Journal of Immunology
3039
have the potential for differentiation in T, B, NK, and dendritic
lineages, but reduced ability to generate myeloid progenitors. Although a sharp boundary for commitment to lymphoid lineages has
not been identified, EBF and Pax5 are essential transcription factors and the latter effectively suppresses alternative differentiation
fates (55). The RAG proteins are required for Ig gene recombination and it has been previously shown that D-J rearrangement
products are first detectable at the CD34⫹CD10⫹CD19⫺ stage
(57, 58). Therefore, the NOD/SCID chimera model could facilitate high-resolution analysis of the earliest categories of human
lymphocyte precursors and investigation of relationships
between them.
Although there are many means to assess lymphopoietic activity
in animal models, methods for evaluation of human marrow are
limited. The chimeric mouse system provides an opportunity to
develop and validate procedures that are useful for human studies.
In this study, we directly compared BrdU incorporation and Ki-67
staining as indices for proliferative activity and found good agreement between the two approaches. This is important in showing
that analysis of single human specimens can be generalized. This
FIGURE 7. Population dynamics and turnover of human B lineage lymphocytes in transplanted NOD/SCID mice. Chimerism was established in
immunodeficient mice within 7 wk of transplantation. At that time, animals were injected and then continuously fed BrdU to label DNA of replicating cells.
Flow cytometry was performed at the indicated intervals with marrow (left panel) and spleen (right panel). A, Half of the large pre-B cells (F, interrupted
line) within bone marrow were labeled within 3 h and were closely followed by pro-B cells (E, fine dashed line). The kinetics of labeling of small pre-B
cells (⽧, coarsely dashed line) was slower and slower still for sIgM⫹ B cells (䡺, solid line). B, Incorporation of BrdU into sIgM⫹ lymphocytes in spleen
is shown and the data best fit a straight line (r2 ⫽ 0.9672). Each point represents results from a single animal.
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FIGURE 6. A substantial pool of quiescent human cells does not establish in transplanted NOD/SCID mice. Marrow was recovered from NOD/SCID
mice 7–10 wk after transplantation. A, Subsets of human cells are gated according to expression of CD34 and low to absent CD38 (left panels). Proliferation
of cells within these two fractions was then evaluated in terms of expression of the Ki-67 Ag (middle and right panels). The comparison is made to similar
cells resolved in samples from normal fetal and adult bone marrow (BM, top and middle rows). B, Grafted mice were sacrificed at the indicated intervals
after BrdU treatment and percentages of CD34⫹CD19⫺ (gating not shown) cells that incorporated the label are given in the upper right quadrants. The
data at each time point are representative of 4 –10 animals evaluated in the same way.
3040
concordance also made it possible for us to compare mitotic activity in chimeric mice to that within freshly isolated fetal and
adult marrow. Percentages of Ki-67⫹ human cells among most B
lineage subsets were remarkably similar in the three environments.
This accords with our finding of similar sized populations of early
and late stage B lineage precursors and presumably means that
human B lymphopoiesis is normal in NOD/SCID marrow. An increased proportion of small pre-B cells in fetal bone marrow expressed Ki67, suggesting that the final stages of B lymphopoiesis
are associated with greater proliferation than during adult life. Ig L
chain gene rearrangement, receptor editing, and selection of the B
cell repertoire all occur during this critical stage and fetal vs adult
differences in cellular expansion merit further study.
A continuous BrdU-labeling protocol was used to assess population dynamics in chimeric bone marrow and spleens. Slightly
more rapid incorporation of BrdU into large pre-B cells than pro-B
cells would be consistent with a short cycle time, and 50% of the
large pre-B cells were BrdU⫹ in just 3 h. Animal studies indicate
that a large fraction of these cells would be in S ⫹ G2 ⫹ M phases
of the cell cycle at any moment in time (59, 60). Small nondividing
pre-B and B cells are sequentially spawned from these replicating
precursors with 50% turnover times of 54 and 105 h, respectively.
We found an 18-h interval between the last dividing cells and
acquisition of sIgM. This compares favorably with a value of 12 h
reported for rats (35). Pulse-chase protocols could be used to obtain more detailed information about the timing and sequence of
progression through the B lymphocyte lineage in bone marrow.
This NOD/SCID system provides an opportunity to experimentally manipulate the bone marrow environment and learn the molecular requirements for maintaining such early lymphocyte precursor populations. We injected engrafted NOD/SCID mice with a
neutralizing Ab to IL-7 and found no influence on human B lymphopoiesis. This is consistent with other findings indicating that
IL-7 is much less important for B lymphocyte formation in humans
than it is in mice (1, 2, 4). Furthermore, administration of IL-7 did
not improve B cell production in NOD/SCID mice and along with
Flt3 ligand actually inhibited emergence of human B cell formation (27). This model might be exploited to learn what other cy-
tokine(s) sustains the survival, proliferation, and differentiation of
human lymphocyte precursors.
Whereas ⬍20% of the B cells in normal human blood express
CD5 (61, 62), most of the B cells recovered from transplanted
mice had this marker. The origin of CD5⫹ B cells has been extensively studied in mice and remains controversial (17, 62). According to one model, CD5⫹ (B1) B cells arise via an independent
differentiation pathway during embryonic life and coexist with
conventional (B2) B cells that are produced within bone marrow
(17). Many human fetal B cells express CD5 and this characteristic
is conceivably intrinsic to cord blood stem cells. However, in preliminary studies, CD5⫹ human B cells were also formed in NOD/
SCID mice transplanted with adult bone marrow stem cells. This
is consistent with findings that CD5⫹ B cells predominate in the
early phase of B cell regeneration after human bone marrow transplantation (63, 64). The CD5⫹ B cells that arose in chimeric NOD/
SCID marrow differed from those in fresh cord blood samples with
respect to CD10 and CD43 Ags (Fig. 5B).
Display of CD5 on B cells has also been said to result from the
specificity and density of surface Ag receptors (19, 20). That is,
low-level recognition of self-Ags in the absence of T cell help may
cause any B lymphocytes to acquire CD5 (18, 62). There are virtually no T cells in the transplanted mice and no information was
available about the specificity of B cell Ag receptors. For those and
other reasons, it was important to determine which Ig VH gene
families are expressed in the NOD/SCID model.
Molecular analysis of the VH transcripts provided several important insights into the model. First, the single-cell PCR studies,
although limited, clearly showed that percentages of VH4-bearing
cells were similar to many previous reports of fetal (40) and cord
blood repertoires (39). That is, the transplanted cells used the full
range of the VH repertoire. This led to a more in-depth study of the
VH4 family.
Analysis of these transcripts resulted in five major conclusions.
1) The utilization of the individual VH4 gene segments was approximately the same as has been reported extensively elsewhere
for both fetal (40) and cord blood (39) human B-lymphocytes. 2)
Human B cells utilized the VH4-39 gene segment less frequently
after exiting the bone marrow. Similarly, upon acquisition of the
CD5 marker, the lymphocytes increased their utilization of VH4-59
and, to a lesser extent, VH4-04. Reciprocally, the use of VH4-61
decreased upon acquisition of CD5. These observations suggest
that the lymphocyte population in these chimeric mice is in a dynamic state, with cells being selected at certain points. 3) HCDR3
lengths as well as the presence of N nucleotides are similar to those
described for human cord blood and term infants (40, 42, 43). This
shows that the TdT expressed by lymphocyte precursors was functional in the murine environment. 4) JH utilization was also typical
of that seen in fetal and cord blood (40, 41). 5) As might be predicted by the absence of T cells in this model, there was no evidence of somatic hypermutation.
Interestingly, VH4-34 was consistently used at the same ratio to
other gene segments by all of the lymphocyte populations studied.
This member of the VH4 gene family is extensively used for B cell
Ag receptors with specificity for RBC and B lymphocyte carbohydrate Ags (65–70). Ongoing studies will reveal whether similar
patterns develop when mice are transplanted with adult human
stem cells and this information may bear on the issue of what
drives CD5 expression. All of these observations indicate that human lymphocyte progenitors rearrange and express Ig genes to
generate immature B cells with a diverse repertoire of Ag receptors. The unique CDR3s obtained for each sequence rule out the
possibility of pauciclonal expansion of small numbers of human B
cells. The results accord with the continuous production of large
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FIGURE 8. Human B cells generated in NOD/SCID mice utilize a diverse repertoire of Ag receptors. Mice were injected with cord blood mononuclear cells pooled from two to three donors. Marrow and spleens were
harvested from two to eight animals 7–10 wk after transplantation, and the
indicated subsets of B lineage lymphocytes were sorted. Utilization of
members of the VH4 family was determined as described in Materials and
Methods. Between 28 and 63 independent sequences were typed for each
of the lymphocyte subsets.
HUMAN B LYMPHOPOIESIS IN NOD/SCID MICE
The Journal of Immunology
Acknowledgments
We are extremely grateful to Viji Dandapani for flow cytometry and cell
sorting and to Stephen Farriester and Amy Fields for sample collection. Dr.
Lisa Borghesi made helpful suggestions on this manuscript and Dr. Fred
Finkelman provided a neutralizing Ab to IL-7. P.W. K. holds the William
H. and Rita Bell Chair in biomedical research.
6.
7.
8.
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26.
27.
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numbers of nondividing lymphocytes from actively proliferating
precursors.
A linear rate of BrdU incorporation into spleen B cells was one
of the most striking findings of this study. Ki-67 staining revealed
almost no dividing cells in that site. Therefore there must be continuous replacement by B cells from the bone marrow. This accords with the immaturity of human B cells with respect to CD24
density, presence of CD38, and display of CD10. Such newly
formed lymphocytes normally represent a small fraction of rodent
spleens, and a majority of the B cells are long-lived (21, 23, 32,
71). There is little information about the normal fate and life span
of CD5⫹ B cells in humans and most of the lymphocytes in chimeric mice expressed this marker. However, peritoneal CD5⫹ B
cells in mice are exceptionally long-lived (72). Furthermore, CD5
expression is common among chronic lymphocytic leukemias and
the presence of somatic hypermutations in their Ig genes would be
consistent with participation in immune responses (62).
Entry of newly formed B cells into the long-lived pool and continued survival normally require expression of an Ag receptor and
one that is not strongly reactive with self-Ags (73, 74). It is theoretically possible that some ligand for the Ag receptor is species
specific and thus lacking in the NOD/SCID environment. More
likely, a species-specific cytokine(s) normally confers a long life
span to B cells by attracting them to nurturing cells in peripheral
lymphoid tissues (75). Finally, T lineage lymphocytes are absent
from the immunodeficient mice and might contribute to life/death
decisions made by newly formed B cells (76, 77).
These findings indicate that in most respects the NOD/SCID
model is representative of B lymphopoiesis in normal human marrow and provide a basis for many other kinds of experimentation.
For example, hormones and cytokines can be manipulated in these
animals to determine their role in human B lymphocyte formation
and potential consequences of therapies can be assessed. However,
the full complement of mature B cells is not generated, suggesting
a role for unknown, species-specific maturation factors. The cellular source of such molecules might be determined by transplantation of mature T, dendritic, NK, or other accessory cells in these
animals. As another approach, IFNs and other species-specific cytokines might be administered. Identification of a human B cell
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diverse repertoire of Ab specificities and suggest new ways to
augment the humoral immune system. NOD/SCID mice might also
provide a controlled environment for comparing the differentiation
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HUMAN B LYMPHOPOIESIS IN NOD/SCID MICE