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Null Mice1
,
* Research Center for Cell Transplantation, and Departments of
Hematology and
Pathology, Tokai University, School of Medicine, Isehara, Kanagawa, Japan; and
Central Institute for Experimental Animal, Kawasaki, Kanagawa, Japan
 |
Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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null (NOD/SCID/cnull) mouse model for human T
lymphopoiesis. When these mice were transplanted with human cord blood
CD34+ cells, the mice reproductively developed human T
cells in their thymus and migrated into peripheral lymphoid organs.
Furthermore, these T cells bear polyclonal TCR-
, and respond not
only to mitogenic stimuli, such as PHA and IL-2, but to allogenic human
cells. These results indicate that functional human T lymphocytes can
be reconstituted from CD34+ cells in
NOD/SCID/cnull mice. This newly developed mouse model is
expected to become a useful tool for the analysis of human T
lymphopoiesis and immune response, and an animal model for studying T
lymphotropic viral infections, such as HIV. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Nonobese diabetic (NOD)/SCID mice have been widely used for the evaluation of human hemopoietic stem cell activity because myeloid and B lymphoid reconstitution can be easily attained in these mice by the transplantation of human HSCs (10, 11). Several reports have suggested that T cells may be able to develop in the thymus of NOD/SCID mice transplanted with human CD34+ cells (12, 13). In particular, van der Loo et al. (13) succeeded in repopulating a mouse thymus with human cells by administering G-CSF and stem cell factor. However, most of the T cells were double-positive (DP) cells, and a functional analysis of these T cells has not been completed.
The abolishment of NK cell activity in NOD/Shi-scid mice by
treatment with antiasialo GM1 antiserum results in a higher degree of
engraftment with human hemopoietic cells (14). On the
basis of this knowledge, NOD/Shi-scid mice were crossed with
mice expressing a form of the IL-2R -chain lacking the cytoplasmic
region, which were reported to have defective NK cells
(15). The resultant NOD/Shi-scid, IL-2R null
(NOD/SCID/cnull) mice have defective T, B, and
NK cell activities.4
In this study, we report that the newly developed
NOD/SCID/cnull mice reproductively develop
human T cells in their thymus, in addition to myeloid and B lymphoid
reconstitution, when transplanted with cord blood (CB)
CD34+ cells. Furthermore, these T cells bear
polyclonal TCR- and respond not only to mitogenic stimuli, such
as PHA and IL-2, but to allogenic human cells. These results indicate
that functional human T lymphocytes can be reconstituted from
CD34+ cells in
NOD/SCID/cnull mice. This newly developed
mouse model is expected to become a useful tool for the analysis of
human T lymphopoiesis and immune response, and as an animal model for
studying T lymphotropic viral infections, such as HIV.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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CB samples were obtained from full-term deliveries, according to institutional guidelines approved by the Tokai University Committee for Clinical Investigation. Mononuclear cells were isolated from the CB samples by Ficoll-Hypaque (Lymphoprep, 1.077 ± 0.001 g/ml; Nycomed, Oslo, Norway) density gradient centrifugation. The cells were washed and suspended in PBS containing 0.2% human serum albumin. The CD34+ cell fraction and CD34- cell fraction were obtained from the Ficoll-separated mononuclear cells using the CD34 Progenitor Cell Isolation Kit (Miltenyi Biotec, Sunnyvale, CA), according to the manufacturer’s directions. The cells were then fractionated on a MACS column type RS using a VarioMACS cell separator (Miltenyi Biotec). The resultant CD34+ cells were >98% pure, and CD3+ cells were not detected on FACS analysis in the sensitivity of 0.1%. CD34- cells were irradiated (15 Gy) and used as carrier cells for CD34+ cell transplantation.
Mice
NOD/Shi-scid, IL-2R null
(NOD/SCID/cnull) mice were generated by
back-cross mating of C57BL/6J-IL-2Rnull mice
to NOD/Shi-scid mice for eight generations.
NOD/Shi-scid (NOD/SCID) and
NOD/SCID/cnull mice4
were obtained from the Central Institute for Experimental Animals
(Kawasaki, Japan) and were maintained in the animal facility of the
Tokai University School of Medicine in microisolator cages with
autoclaved food and water. The mice were irradiated at 7–9 wk of age
with 250 cGy 137Cs x-rays, and thereafter
received acidified water containing 1.1g/L neomycin sulfate and 131
mg/L polymyxin B sulfate (Sigma-Aldrich, St. Louis, MO). The following
day, 8 x 104 to 2 x
105 CD34+ CB cells were
injected i.v., along with 106 irradiated (15 Gy)
carrier cells. A total of 5 NOD/SCID and 27
NOD/SCID/cnull mice were used in 6 independent
experiments.
Flow cytometric analysis
Six to nineteen weeks after transplantation, the mice were anesthetized with ethyl ether, and peripheral blood was sampled from the retroorbital sinus. At the time of sacrifice, the bone marrow, spleen, and thymus were collected and stored in PBS containing 0.2% human serum albumin. These tissues were teased apart and passed through a nylon filter to remove debris. Samples were prepared as single cell suspensions in PBS. Cells were stained with mAbs to human leukocyte differentiation Ags: FITC-conjugated anti-human CD1a (NA1/34; DAKO, Glostrup, Denmark), CD3 (UCHT1), CD34 (581), CD41 (P2), glycophorin A (11E4B-7-6) (all Coulter/Immunotech, Marseille Cedex, France), CD4 (Leu-3a), CD14 (Leu-M3), CD19 (SJ25C1), CD33 (Leu-M9), CD45RA (Leu-18), and CD56 (NCAM16.2) (all BD Biosciences, San Jose, CA); PE-conjugated anti-human CD8 (Leu-20), CD11c (Leu-M5), and CD45RO (Leu-45RO) (all BD Biosciences); and APC-conjugated anti-human CD45 (J.33; Coulter/Immunotech). A FACS analysis was conducted by three- or four-color flow cytometric analysis using a FACSCalibur or FACSVantage (BD Biosciences). Quadrants were set to include at least 97% of the isotype-negative cells. Dead cells stained by propidium iodide were excluded from the analysis.
Immunohistochemistry
Nineteen weeks after transplantation,
NOD/SCID/cnull mice thymuses were frozen in
OCT embedding medium (Sakura Finetechnical, Tokyo, Japan). Sections
were air dried and fixed with acetone. Fixed samples were stained with
mAbs to specific surface Ags. Biotinylated mAbs against human CD3
(UCHT1; Coulter/Immunotech) and human CD80 (BB1) and isotype controls
(BD Biosciences). Staining was shown after incubation with
HRP-conjugated streptavidin (DAKO), followed by diaminobenzidine
substrate. All samples were counterstained with H&E. C57BL/6 mouse
thymuses were used as negative controls.
Analysis of TCR V repertoires of human T lymphocytes
For the FACS analysis of the expression of TCR V-chains, the
TCR V repertoire kit (Coulter/Immunotech) was used according to the
manufacturer’s protocol. In brief, cells from the mouse spleen were
stained with anti-human mAbs against ECD-CD45 and APC-CD3 as well
as with V-specific anti-human mAbs (conjugated with FITC and PE)
against TCR.
Proliferation assay
The total splenocyte population was cultured in RPMI 1640 medium
containing 20% human serum with PHA (Life Technologies, Grand Island,
NY) or 10 ng/ml human rIL-2 (R&D Systems, Minneapolis MN) in
round-bottom microtiter plates. The proliferation of human T cells in
NOD/SCID/cnull mice was also tested in a mixed
leukocyte reaction assay by stimulating 6 x
104 splenocytes with 6 x
104 mitomycin C (Kyowa Hakko Kogyo, Tokyo,
Japan)-treated EBV-transformed B-lymphoblastoid cell line (B-LCL) cells
(kindly donated by M. Hagihara, Tokai University School of Medicine)
and allogeneic human PBLs in round-bottom microtiter plates.
Splenocytes from BALB/c mice (H-2d; purchased from Charles River,
Yokohama, Japan) and C3H mice (H-2k; Charles River) were also used as
stimulators. These cultures were incubated for 4–6 days at 37°C in a
humidified atmosphere of 5% CO2. Proliferating splenocytes
were stained with anti-human APC-CD3, FITC-CD19, and PerCP-CD45,
and then analyzed using a FACSCalibur. During the last 6 h, 1
µCi [3H]thymidine was added to the culture.
Subsequently, the cultures were harvested onto fiberglass filters, and
[3H]thymidine incorporation was determined
using liquid scintillation spectroscopy.
Statistical analysis
Results are expressed as individual data or as the mean ± SD. Statistical comparisons were performed according to Mann-Whitney U test. The two-sided p value was determined testing the null hypothesis that the two population medians are equal. Values of p <0.05 were considered significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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cnull
mice compared with NOD/SCID mice
To compare the engraftment and multilineage reconstitution of the
human hemolymphoid system, 5 NOD/SCID and 27
NOD/SCID/cnull mice were transplanted with
8 x 104 to 2 x
105 CD34+ CB cells. Each
mouse received CB cells derived from different single donor. In these
transplanted CD34+ cells, none of
CD3+ mature T cells were detected by FACS
analysis (Fig. 1
Ab).
Peripheral blood was collected at various intervals up to 16 wk
(n = 17) to 19 wk (n = 10) and analyzed
by flow cytometry for the presence of human cells expressing the
leukocyte common Ag CD45 and the CD3 or CD19 lymphoid lineage markers.
Six weeks after transplantation,
CD45+CD19+ human B cells
(Fig. 1Bc), but not
CD45+CD3+ human T cells
(Fig. 1Bb), were detected in both NOD/SCID and
NOD/SCID/cnull mice. Human
CD3+ T cells, however, began to emerge at 13 wk,
then increased up to 67.5% (10.85% to 67.5%) (n =
10) of the CD45+ cells at 19 wk in all of the
NOD/SCID/cnull mice (Fig. 1Bb), but
not in the NOD/SCID mice (data not shown). All of the mice sustained
stable chimerism for human CD45+ cells over 19
wk, but the rate was much higher in the
NOD/SCID/cnull mice (6.8 ± 2.7%
(n = 5) in NOD/SCID mice and 54.69 ± 24.1%
(n = 10) in NOD/SCID/cnull
mice (p < 0.01)). Representative profiles of
CD3+ cell kinetics from the same individual
NOD/SCID/cnull mouse were shown in Fig. 1c.
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cnull mice at 19 wk
(p < 0.01). Multilineage differentiation of
human SCID-repopulating cell was observed in the bone marrow of the
transplanted NOD/SCID and NOD/SCID/cnull mice,
including lymphoid
CD45+CD19+ cells and
myeloid CD45+CD33+ cells as
well as primitive CD34+ cells. Cells recovered
from the bone marrow of these mice could also differentiate into
lymphoid CD45+CD56+ NK
cells (data not shown). In the NOD/SCID/cnull
mice, 1.22 ± 1.9% (n = 10) of
CD45+CD3+ cells were
detected in the bone marrow, but no
CD45+CD3+ cells were
detected in the NOD/SCID mice.
Characterization of the T lymphocytes reconstituted from CB
CD34+ cells in the NOD/SCID/cnull mice
To further characterize the T lymphocytes emerging in the
NOD/SCID/cnull mice, cells harvested from the
thymus, spleen, and peripheral blood were analyzed for other T cell
markers in all mice (n =
27). Representative data were shown in Figs. 2 and 3. Most thymocytes in the
NOD/SCID/cnull mice were human
CD3+ cells (Fig. 2a).
CD19+ B cells and
CD1a+CD11c+ dendritic cells
(DCs) were detected in a CD3- cell population
(not shown). Histologic examination revealed that thymus of
NOD/SCID/cnull mice displayed a frail, but a
typical thymic construction that defined cortex and medulla densely
(Fig. 3A). The serial sections of this region were further
analyzed by immunohistochemistry. The region of medulla was strongly
stained by anti-human CD3 mAb (Fig. 3B).
CD80+ DC-like cells were also detected in the
same medulla regions (Fig. 3C). These results demonstrated
that NOD/SCID/cnull mouse thymus was mainly
constructed with CD3+ human T cells and,
otherwise, was constructed with CD19+ human B
cells and CD80+ human DC-like APCs. The
CD3+ cells were comprised of both CD4/CD8 DP and
SP subsets at 19 wk after transplantation (Fig. 2b). Further
phenotypic analysis confirmed that the human CD3+
thymocytes expressed TCR- (not shown) and exhibited a decreased
level of CD1a expression (Fig. 2d). This phenotype is
consistent with that of terminally differentiated thymocytes, further
evidence of a full thymic T cell development program. These data
demonstrate that human T cell progenitors derived from
CD34+ cells can proliferate and differentiate
into mature T cells in the NOD/SCID/cnull mice
thymic microenvironment. In the spleen and peripheral blood, most of
the CD3+ T cells expressed CD4/CD8, TCR-,
and CD45RA on their surfaces (Fig. 2, e–l). A few T cells
expressing TCR-
and CD45 RO were detected in the spleen and
peripheral blood. These data suggest that most of the T cells were
naive type T cells. To further examine the clonality of human T
cells in NOD/SCID/cnull mice, TCR V
expression was analyzed by flow cytometry. As shown in Fig. 4, all V TCR repertoires were used in
the T cells of all four mice analyzed. The usage of V repertoires in
every mouse was similar to that observed in human T cells.
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cnull mice
To confirm that this NOD/SCID/cnull mice
system could indeed produce the whole spectrum of mature T cells, we
cultured unseparated splenocytes with PHA or IL-2. In all cases
(n = 8), the splenocytes grew vigorously in response to
the stimuli, demonstrating a functional response (Fig. 5A, p <
0.01). PHA-responding proliferating splenocytes were analyzed by flow
cytometry. As shown in Fig. 5B, the PHA-responding cells
were mainly human T cells. Furthermore, splenocytes obtained from the
NOD/SCID/cnull mice spleen proliferated
vigorously when stimulated with any third-party B-LCL tested, including
normal donor-derived PBL (Fig. 5C, p <
0.01). In contrast, the cells did not respond to murine cells bearing
H-2d and H-2k. These data show that the CB CD34+
cells can generate polyclonal T cells that are functional in the
context of a strong proliferative response to mitogens, IL-2, and
alloantigens in NOD/SCID/cnull mice.
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T cell repertoire
shown in Fig. 4
, indicate the absence of GVHD. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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+CD4+
or CD8+ was compromised in these systems
(9, 17). Res et al. (18) indicated that
murine thymic epithelial cells were not capable of inducing
functionally mature human T cells. In this study, we have demonstrated
that mature T lymphocytes can develop from human CB
CD34+ cells in the thymus of
NOD/SCID/cnull mice. The resultant T cells
were polyclonal and responded to mitogenic, cytokine, and allogeneic
proliferation stimuli.
Organ-specific T cell development was observed in
NOD/SCID/cnull mice. The phenotype of human
CD3+ T cells detected in the spleen and
peripheral blood was that of mature SP T cells, whereas DP
CD3+ cells with CD45RA and CD1a were detected in
the thymus. Furthermore, mature T cells began to emerge at 12 wk after
transplantation, whereas DP cells were already detected in the thymus
at 8 wk after transplantation (our unpublished data). These temporal
and spatial patterns of T cell distribution imply that immature human T
cells were generated in the thymus and then preferentially
developed and/or colonized in the spleen of
NOD/SCID/cnull mice.
The contamination of mature T cells in the transplanted cells was
unlikely because of: 1) undetectable CD3+ cells
in the transplanted cells, as shown in Fig. 1A; 2)
polyclonal V repertoires, as shown in Fig. 4; and 3) the absence of
GVHD. However, the clonal analysis in gene-marking study would provide
the definitive answer to the origin of mature T cells in mice.
The ability of the T cells to respond to allogenic stimulation
indicates that positive selection has occurred in the mice. The degree
of T cell response to third party cells in the
NOD/SCID/cnull mice was the same as that of T
cells from a normal volunteer. The MLC results indicate that the
development of T cells is restricted by human MHC. Because human
CD19+ B and CD80+ DC were
found in the thymus, we believe that these cells may induce thymocyte
selection (19, 20, 21, 22). Another possibility is that the mouse
epithelial cells support human T cell education, because it has been
reported that human CD8 interacts with the 3 domain of murine MHC
class I and that human CD4 interacts with murine MHC class II molecules
(23, 24, 25). If the latter possibility is the case, the
alloantigen would be presented in the context of murine MHC. The
precise mechanism of positive selection in this system is unknown, and
further studies are required.
The T cells that developed in the mice were not responsive to murine cells bearing either H-2d or H-2k during the MLC assay. One possible reason for this result is that the murine class I and class II molecules are poorly reactive to human cells (17). However, because PBLs transplanted into SCID mice cause GVHD (26, 27, 28, 29), the absence of GVHD indicates that the T cells are tolerant to the murine cells. The T cells were also not reactive to autologous HLA Ags from other lineages of human cells derived from CD34+ cells, suggesting that the T cells were tolerant to autologous Ags, possibly those presented by B cells or DCs (30). Therefore, negative selection may have resulted in the clonal deletion of cells reactive to both autologous human and murine Ags.
There are several possible explanations for the differences in T cell
maturation in the thymus of NOD/SCID and
NOD/SCID/cnull mice. Human
CD34+ cells do not always migrate to the thymus
when they are implanted into NOD/SCID mice. When G-CSF was
administered, CD3+CD4+
cells were found in the thymus of highly engrafted NOD/SCID mice,
suggesting the existence of barrier between certain species that
prevents the repopulation of T cell progenitors in the thymus
(13). The incidence of human cells in the PB of
NOD/SCID/cnull mice was 7.8 times higher than
that of NOD/SCID mice, whereas that in the bone marrow of
NOD/SCID/cnull mice was only 1.7 times higher
than that of NOD/SCID mice. This indicates a higher rate of cell
migration from bone marrow to peripheral blood and possibly to the
thymus in NOD/SCID/cnull mice, compared with
NOD/SCID mice. As a result, the thymus of
NOD/SCID/cnull mice may be reconstituted with
T cell-educating cells, such as DCs and B cells, derived from human
CD34+ cells, creating an environment for T cell
maturation. Further investigation is necessary to determine the
mechanism responsible for reconstitution of T cells in
NOD/SCID/cnull mice. This mouse system is an
ideal model for the study of positive selection (MHC restriction) and
negative selection (self-tolerance) in developing human T cells.
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Acknowledgments |
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cnull mice, Hideyuki Matsuzawa for technical
assistance, Johbu Itoh and Yoshiko Itoh for immunohistochemical
analysis, Jeffrey Miller (University of Minnesota) for critical
reading, Shizuko Imai for secretarial work, and the members of Tokai
Cord Blood Bank for their assistance. |
Footnotes |
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2 Address correspondence and reprint requests to Dr. Kiyoshi Ando, Department of Hematology, Tokai University School of Medicine, Bohseidai, Isehara, Kanagawa, Japan. E-mail address: andok{at}keyaki.cc.u-tokai.ac.jp
3 Abbreviations used in this paper: HSC, hemopoietic stem cell; B-LCL, EBV-transformed B-lymphoblastoid cell line; CB, cord blood; DC, dendritic cell; DP, double positive; FTOC, fetal thymic organ culture; GVHD, graft-vs-host disease; NOD, nonobese diabetic; SP, single positive; SRC, SCID-repopulating cell.
4 M. Ito, K. Kobayashi, K. Suzue, M. Kawahata, K. Hioki, Y. Ueyama, T. Koyanagi, K. Sugamura, K. Tsuji, H. Hiramatsu, T. Heike, and T. Nakahata. D-NOD/Shi-scid, IL-2R null mice: a novel excellent recipient mouse for engraftment of human cells. Submitted for publication.
Received for publication February 1, 2002. Accepted for publication May 1, 2002.
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S. Samira, C. Ferrand, A. Peled, A. Nagler, Y. Tovbin, H. Ben-Hur, N. Taylor, A. Globerson, and T. Lapidot Tumor Necrosis Factor Promotes Human T-Cell Development in Nonobese Diabetic/Severe Combined Immunodeficient Mice Stem Cells, November 1, 2004; 22(6): 1085 - 1100. [Abstract] [Full Text] [PDF]
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N. Kambe, H. Hiramatsu, M. Shimonaka, H. Fujino, R. Nishikomori, T. Heike, M. Ito, K. Kobayashi, Y. Ueyama, N. Matsuyoshi, et al. Development of both human connective tissue-type and mucosal-type mast cells in mice from hematopoietic stem cells with identical distribution pattern to human body Blood, February 1, 2004; 103(3): 860 - 867. [Abstract] [Full Text] [PDF]
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S. Rutella, G. Bonanno, M. Marone, D. de Ritis, A. Mariotti, M. T. Voso, G. Scambia, S. Mancuso, G. Leone, and L. Pierelli Identification of a Novel Subpopulation of Human Cord Blood CD34-CD133-CD7-CD45+Lineage- Cells Capable of Lymphoid/NK Cell Differentiation After In Vitro Exposure to IL-15 J. Immunol., September 15, 2003; 171(6): 2977 - 2988. [Abstract] [Full Text] [PDF]
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