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HEMATOPOIESIS
From the Department of Clinical Chemistry,
Microbiology, and Immunology and the Department of Hematology, Ghent
University Hospital, Belgium; the Division of Clinical Onco-Immunology,
Ludwig Institute for Cancer Research, University Hospital, Lausanne,
Switzerland; the Department of Immunology, University Hospital
Rotterdam, The Netherlands; and BTC Oost-Vlaanderen, Ghent, Belgium.
The NOD-LtSZ scid/scid (NOD/SCID) repopulation assay is the
criterion for the study of self-renewal and multilineage
differentiation of human hematopoietic stem cells. An important
shortcoming of this model is the reported absence of T-cell
development. We studied this aspect of the model and investigated how
it could be optimized to support T-cell development. Occasionally,
low-grade thymic engraftment was observed in NOD/SCID mice or
Rag2 The NOD/SCID model, which sustains proliferation
and engraftment of intravenously injected human hematopoietic stem
cells (HSCs), mimics engraftment after clinical transplantation. An important shortcoming of this model is that only exceptional
thymopoiesis is seen.1-5 Therefore, the
multipotency of stem cells cannot be completely evaluated in this
model, nor can this model be used to study T-cell reconstitution after
stem cell transplantation.
Several models do exist to study the T-cell potential of HSCs. First,
the SCID-Hu model enables the study of T-cell development from
CD34+ HSCs after direct injection into a human thymus
established from fetal material several weeks earlier.6,7
Second, fetal thymus organ culture supports T-cell development from
CD34+ HSCs that were introduced in fetal thymic lobes from
immunodeficient mice.8 These models require fetal material
or they exclusively generate T cells. Hence, functional aspects of T
cells cannot be studied in these models.
To address this issue, strategies have been introduced to study T-cell
development in the NOD/SCID model. Robin et al9 have
identified T-cell progenitors in the bone marrow of NOD/SCID mice
injected with CD34+38lo and
CD34+38+ cells from human adult bone marrow and
umbilical cord blood (UCB) by their capacity to form T cells when
introduced into fetal thymus organ culture. Crisa et al4
transplanted human fetal thymus under the kidney capsule of NOD/SCID
mice that were injected with T- and B-cell-depleted human UCB 4 to 6 weeks earlier. They observed T-cell development from injected human UCB
cells in the human thymus graft. These models are time consuming and
technically cumbersome.
When studying T-cell development in the NOD/SCID repopulation model, we
could confirm that thymopoiesis did not occur in most experiments.
However, a low percentage of mice did show evidence of thymopoiesis. We
wondered whether we could improve thymopoiesis by blocking the natural
killer (NK) cell activity in these mice. We injected intraperitoneally
TM- Monoclonal antibodies
Mice
Cell sources UCB was obtained from full-term healthy newborns, and mononuclear cells were isolated within 24 hours after collection using a Lymphoprep density gradient (Nycomed Pharma, Oslo, Norway). UCB samples were obtained and used according to the guidelines of the Medical Ethical Commission of the Ghent University Hospital. Unless used fresh, cells were resuspended in 90% heat-inactivated fetal calf serum (Life Technologies, Paisley, Scotland)-10% dimethyl sulfoxide (Serva, Heidelberg, Germany) and were frozen in liquid nitrogen until use. The mononuclear cell fraction contained 53% ± 19% (n = 8) CD45+ cells (absolute number, 5.4 ± 2.3 × 106 CD45+ cells). The mononuclear cell fraction was stained with mouse anti-human CD3 mAb for immunomagnetic depletion using sheep anti-mouse immunoglobulin-coated beads (Dynabeads; Dynal AS, Oslo, Norway) with a cell-bead ratio of 1:4. After this procedure the percentage of CD3+ cells within the CD45+ cell fraction decreased 1 log, resulting in 0.63% ± 0.38% CD3+ cells, representing absolute numbers of 2.8 ± 1.9 × 104. The average percentage of CD34+ cells after T-cell depletion was 2.3% ± 1% of the CD45+ cells. The total number of injected CD34+ cells was 11.7 ± 4.6 × 104. In one experiment CD34+CD3 sorted UCB cells were
used. For this experiment, UCB cells were enriched in CD34+
cell content using magnetic cell sorting (MACS) (Miltenyi
Biotec, Bergisch Gladbach, Germany), and then
CD34+CD3 cells were sorted. In experiments
for T-cell receptor excision circles (TREC) analysis and
TCR-V repertoire analysis, MACS (Miltenyi Biotec) selected
CD34+ cells were used. On average we injected per mouse
10.6 ± 9.5 × 104 cells, containing 97% ± 2%
CD34+ cells (resulting in absolute numbers of
10.3 ± 9.2 × 104 CD34+ cells) and
containing 0.25% ± 0.15% CD3+ cells (262 ± 145
CD3+ cells).
NOD/SCID repopulation assay Six- to 8-week-old mice were given a sublethal dose of whole-body irradiation (350 cGy, 12-15 cGy/min) using a Cobalt radiation source and were injected intraperitoneally with 200 µg TM-1, an antibody functionally blocking the mouse IL-2R chain.
Twenty-four hours later the mice were injected intravenously (in the
tail vein) with human UCB cells. Eight to 15 weeks after injection, the
mice were killed and peripheral blood, thymus, liver, spleen, mesenteric lymph nodes, and both femora (in some experiments together with both tibia bones) were used for analysis. Cell suspensions from
these organs were filtered through a 70-µm cell strainer. Red blood
cells were lysed using hypotonic lysing buffer. Cells were counted,
cell viability was checked (greater than 85%), and, after blocking the
Fc-receptor, cells were labeled with mAbs and analyzed on a flow cytometer.
Flow cytometry and cell sorting Cells were analyzed or sorted on a FACSCalibur (BDIS) or a FACS Vantage (BDIS), respectively, each equipped with an argon-ion laser tuned at 488 nm and a red-diode laser tuned at 635 nm. For analysis of viable human cells, gates were set on the propidium iodide (PI)-negative, human CD45+ cells. Data acquisition and analysis were performed with CellQuest software (BDIS).Proliferation assay A total of 5 × 104 cells/well harvested from murine thymus or spleen were incubated in 96-well, round-bottomed microtiter plates in the presence of 2 µg/mL PHA (Wellcome Diagnostics, Beckenham, United Kingdom), 50 U/mL recombinant human IL-2 (kindly provided by M. Gately, Hoffmann-La Roche, Nutley, NJ) (first week) and feeders (irradiated [2500 cGy] human peripheral blood mononuclear cells, 106/well). Two weeks later cells were restimulated in the presence of PHA and fresh feeders for another week.Quantitative polymerase chain reaction of TRECs Quantification of signal-joint TRECs in sorted human CD3+ populations from repopulated mice was performed by real-time quantitative polymerase chain reaction (PCR) with the 5' nuclease (Taqman) assay and an ABI 7700 system (Perkin-Elmer, Foster City, CA). As previously described,13 cells were lysed in 100 mg/L proteinase K (Roche Molecular Diagnostics, Rotkreuz, Switzerland) for 2 hours at 56°C, then for 15 minutes at 95°C. Each PCR reaction was performed in a 25 µL solution containing 5 µL cell lysate, 12.5 µL Taqman Universal Master Mix including AmpliTaq Gold (Applied Biosystems, Rotkreuz, Switzerland), 500 nM of each primer (signal-joint 5' forward, CAC ATC CCT TTC AAC CAT GCT; signal-joint 3' reverse, GCC AGC TGC AGG GTT TAG G) and 125 nM probe (FAM-ACA CCT CTG GTT TTT GTA AAG GTG CCC ACT-TAMRA) in a final volume of 25 µL. PCR conditions consisted of 1 cycle of 2 minutes at 50°C and 1 cycle of 10 minutes at 95°C, followed by 40 cycles of 30 seconds at 95°C and 1 minute at 65°C. Tenfold serial dilutions ranging from 107 to 101 copies of signal-joint internal standard, kindly provided by Dr Daniel Douek (Department of Internal Medicine, University of Texas, Southwestern Medical Center), were tested in quadruplicate in each PCR experiment. A standard curve with a linear range across 7 log DNA concentration was created that allowed calculation of the number of TRECs in a given cell population. Thus, the lowest limit of quantitation was considered to be 10 copies of the target sequence. In all PCR assays, the correlation coefficient of the curve was 0.995 or greater, whereas the slope varied between 3.54 and
3.73. TREC levels were quantified by 6 PCR replicates.
Stimulation and cytokine production measurement A total of 5 × 104 cells/well were incubated in 96-well, flat-bottomed microtiter plates and were stimulated with 10 µg/mL anti-CD3 mAb (OKT3; ATCC) and 10 ng/mL TPA (Sigma Chemical, St Louis, MO) or 10 µg/mL concanavalin A (ConA; Serva, Heidelberg, Germany) for 24 hours. For CD3 stimulation, plates were coated with 100 µL/well of 10 µg/mL purified OKT3 in phosphate-buffered saline (1 hour incubation at 37°C) and then were washed 3 times with phosphate-buffered saline. After 24 hours of stimulation, cultures were spun down, and supernatant was harvested from each well and kept frozen at20°C until the determination of cytokine content. Each
stimulation was performed in duplicate. The content of IL-2, IL-4, and
interferon- (IFN-) was determined using ELISA tests IL-2 EASIA,
IL-4 EASIA, and IFN- EASIA, respectively (BioSource, Nivelle,
Belgium). Tests were performed according to the manufacturer's instructions.
TCR-V analysis, RNA was isolated from sorted
CD45+ cell fractions from thymus and total bone marrow
using TRIzol (Life Technologies). After oligo dT annealing, total RNA
was subsequently reverse transcribed using Superscript II RT enzyme
(Life Technologies) in the presence of dNTPs and RNAguard (Amersham
Pharmacia Biotech, Uppsala, Sweden). PCR amplification and heteroduplex
analysis were performed as described before.14
Statistical analysis Statistical analysis was performed using SPSS for Windows software, version 9.0. Results are expressed as the mean ± SE. Differences were evaluated with Mann-Whitney U test where appropriate. Statistical significance was assumed for P < .05.
T-cell development in NOD/SCID mice T-cell development is reported to occur exceptionally in NOD/SCID mice injected with a human stem cell source. To quantify the frequency of T-cell development, we injected human CD3 depleted UCB in NOD/SCID mice irradiated with 350 cGy at least 24 hours, and at most 48 hours, before. Twelve to 15 weeks after injection, the mice were killed and their organs were analyzed for the presence of human cells. We focused on human T-cell development in the murine thymus. We indeed found some mice (5 of 28) with a repopulated thymus, defined by the presence of CD4+CD8+ DP cells in the thymus, though the absolute number of human thymocytes was low. A typical dot of such a poorly reconstituted T-cell-containing thymus is shown in Figure 1.
Influence of IL-2R 1, an antibody blocking the murine
IL-2R, to irradiated NOD/SCID mice before injection of human cells.
Second, we used irradiated
Rag2/c/ mice, which lack
mature B, T, and NK cells,16 as hosts for human cells
instead of NOD/SCID mice. The frequencies of successful thymus
repopulation achieved in both adapted repopulation models are shown in
Table 1. Addition of TM-1 treatment
increased significantly, from 18% to 56%, the frequency of mice with
successful thymus repopulation with human DP cells. This suggests that
NK cells indeed negatively influence the success rate of thymopoiesis. However, thymus repopulation observed in
Rag2/c/ mice was similar
to that in NOD/SCID mice. This indicates that besides eliminating T-
and B-cell function, the blocking of NK activity is insufficient for
increased human lymphopoiesis in the murine thymus.
Increased chimerism after TM-
Although human cells could be observed in the bone marrow within hours of transplantation, thymopoiesis was seldom seen before 2 weeks after transplantation. Thymus repopulation increased up to week 15 (data not shown). The time delay in thymopoiesis and repopulation kinetics is compatible with the notion that stem cells from the bone marrow seed to the thymus only after homing to the mouse bone marrow rather than at the time of inoculation. Phenotypical analysis and molecular V terminal differentiation stage cannot
develop17 or can develop only in low numbers after extended
culture periods.8 Therefore, we analyzed the terminal
differentiation stages more closely in our model. Phenotypically, all
consecutive developmental stages were present:
CD4+CD8+, CD3+CD69+,
CD3+CD27+, and
CD3+CD1 cells (which are
TCR- +) (Figure 3), suggesting that terminal
differentiation occurred in these murine thymi. The
CD3+CD1 cells were present in sufficient
numbers to perform functional studies. The number of cells in a
repopulated thymus was on average 4.7 ± 3.7 × 106
(n = 22), of which 77% ± 28% were human CD45+.
CD3+CD1 mature cells represent on average
4.6% ± 2.3% of the human thymocytes, which are at least
105 cells. The clonality of the thymus repopulation was
addressed by molecular V analysis. RNA was isolated from sorted
human CD45+ fractions from a repopulated thymus. V-C
transcripts were amplified by reverse transcription RT-PCR. As shown
in Figure 4A, rearrangements were found
across most human V families. In addition, a clear polyclonal
pattern (smear) was seen in the more prominent V families in
heteroduplex analysis, confirming the presence of a broad V repertoire.
Human T cells in the periphery After injection of CD3-depleted UCB cells, human cells could be detected in bone marrow, spleen, and peripheral blood within weeks after transplantation. Occasionally, contaminating T cells that were injected in a few mice expanded and could be transiently measured in the peripheral blood up to a few weeks after injection, after which these cells disappeared. Eight to 15 weeks after transplantation, T cells reappeared in the peripheral blood (Table 2). However, at this time T cells were observed only in mice with repopulated thymi. This observation, together with the delay in appearance of T cells in the periphery, favors the hypothesis that these cells were derived from the thymus. To strengthen this hypothesis, rigorously sorted CD34+CD3 UCB cells were injected into
NOD/SCID mice (on reanalysis, T-cell contamination was less than
0.1%). These mice were analyzed 12 weeks later for the presence of
peripheral T cells. Three of 5 mice had human double-positive cells in
the thymus. Two of these successfully thymic repopulated mice
selectively carried human T cells in the peripheral blood. This
strongly argues against the expansion of contaminating CD3+
cells and indicates that the observed T cells were generated de novo in
these animals (Figure 5).
To investigate whether these peripheral CD3+ cells could be
activated in vivo and could mount an immune reaction, phenotypic analysis for naive and memory cells was performed. Some animals (6 of
13) contained low percentages of human cells that were mainly CD45RA+ (Figure 6A), similar
to the phenotype of thymic emigrants. However, in some mice (7 of 13),
CD45RO+ cells were abundantly observed, of which both
CD27
To investigate whether the human peripheral T cells were functional,
spleen cells were cultured in the presence of PHA + IL-2. From the
3 spleens cultured, a human T-cell line was generated. On stimulation
with OKT3 + TPA or ConA, significant levels of IL-2, IL-4, and
IFN-
TREC analysis of thymocytes and peripheral T cells To confirm that the repopulation of the thymus results from the injected CD34+ cells, we determined the number of TRECs in the sorted human CD45+CD3+CD27/69+ cells from the thymus. This CD45+CD3+CD27/69+ population was sorted from the thymus, and TREC levels were determined. TREC levels were relatively high in the thymuson average, 97 ± 17 TRECs per
100 cells.
To further exclude that peripheral human T cells were the result of
expansion of injected contaminating CD3+ cells, TREC levels
were determined on these cells. We sorted CD3+CD45RA+ and
CD3+CD45RA
The NOD/SCID model is widely accepted as the best-suited model to
study self-renewal and multilineage differentiation capacity of human
HSCs. A major drawback of the model is the inability to study T-cell
development from HSCs because the frequency of thymus repopulation is
extremely low. Here we show that by pretreatment of the NOD/SCID mice
with irradiation and TM- NOD/SCID mice are relatively deficient in NK cell activity, but the
administration of anti-asialo GM-1 or TM- Phenotypic analysis of the human thymocytes shows that all maturational
stages are present, from the most immature to the terminally mature
stage. The consecutive stages in maturation were described in the
neonatal thymus and the SCID-Hu(liv/thy) model by our
laboratory.23,24 We showed that functionally immature CD3+CD69+CD27 Vanhecke et al23 have shown that the capacity to proliferate on IL-2 after stimulation is absent in the most immature thymocytes and is acquired gradually on maturation. Clonal expansion and capacity to respond to stimulation with the production of cytokines are also acquired during the final stages of maturation. In our model, human cells harvested from thymus and spleen could be clonally expanded, and they produced cytokines on stimulation, proving that the phenotypically identified final maturation stages are also functionally mature. Through molecular V These T cells resided in the periphery in the presence of human and
mouse allophycocyanin. We focused our analysis on signs of T-cell
activation. In 6 of 13 mice, we found mainly
CD3+CD45RA+ cells and a small number of
CD3+CD45RO+ cells in the periphery. In
addition, in the SCID-hu model, in which T cells survive only for a few
hours in the periphery, a similar pattern is seen.24,25
Although we could not fully exclude that the CD45RO+ cells
observed contaminated T cells instead of thymic emigrants, 7 of 13 mice
had high percentages of CD45RO+ cells and virtually no
CD45RA+ cells. Moreover, the number of peripheral T cells
was much higher in the mice with primarily CD45RO+ cells in
the periphery. Together these data suggest that T cells generated in
the mouse thymus were functional in these mice and were probably
activated by a foreign antigen in the context of mouse or human antigen
presenting cells. The difference in V Because T-cell depletion of the injected cells was incomplete in most
of these experiments, we could not formally exclude that the peripheral
T cells observed 10 to 15 weeks after injection were not derived from a
small contamination of CD3+ cells still present after CD3
depletion. It has been reported that mature circulating T cells were
never detected in SCID mice, regardless of whether they were
reconstituted with whole CB (n = 50) or purified CD34+
cells (n = 20). This finding provides arguments against survival and
proliferation of injected T cells.4 We also addressed this question experimentally by injecting highly purified
CD34+CD3 In conclusion, blocking the IL-2R This regimen holds great promise for a more complete experimental approach of stem cell reconstitution capacity after transplantation of fresh or long-term cultured HSCs and for the evaluation of drugs to boost human T-cell reconstitution.
We thank An De Creus and Tom Taghon for stimulating discussions;
Achiel Moerman and Caroline Collier for animal care; Christian De
Boever for artwork; Ingrid Wolvers-Tettero for expert technical assistance in the V
Submitted June 20, 2001; accepted October 26, 2001.
Supported by grants from the Gezamenlijk Overlegde Actie, Ghent University, Belgium; the Fund for Scientific Research Flanders, Belgium; and the Flanders Interuniversity Institute for Biotechnology, Belgium. T. K. is a research assistant of the Fund for Scientific Research Flanders, Belgium. A.Z. was supported by grant Zi685/1-1 from the Deutsche Forschungsgemeinschaft and M.J.P. was supported by grant KFS 633-2-1998 from the Swiss Cancer League.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Tessa Kerre, Department of Clinical Chemistry, Microbiology, and Immunology, Ghent University Hospital, 4BlokA, De Pintelaan 185, B-9000 Ghent, Belgium; e-mail: tessa.kerre{at}rug.ac.be.
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X. Wang, S. Ge, G. McNamara, Q.-L. Hao, G. M. Crooks, and J. A. Nolta Albumin-expressing hepatocyte-like cells develop in the livers of immune-deficient mice that received transplants of highly purified human hematopoietic stem cells Blood, May 15, 2003; 101(10): 4201 - 4208. [Abstract] [Full Text] [PDF]
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M. De Smedt, K. Reynvoet, T. Kerre, T. Taghon, B. Verhasselt, B. Vandekerckhove, G. Leclercq, and J. Plum Active Form of Notch Imposes T Cell Fate in Human Progenitor Cells J. Immunol., September 15, 2002; 169(6): 3021 - 3029. [Abstract] [Full Text] [PDF]
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| Copyright © 2002 by American Society of Hematology Online ISSN: 1528-0020 | |||||||||
Top
Abstract
Introduction
p9), CD19
(4G7), CD27 (M-T271), CD34 (8G12), CD38 (HB7), CD45 (2D1), CD45RA
(HI100), CD45RO (UCHL1), CD56 (MY31), CD62L (Dreg 56), CD69 (FN50), and
HLA DR (L243) from Becton Dickinson Immunocytometry Systems (BDIS, San
Jose, CA); and Diversi-T (panel of mAbs to human T-cell receptor
[TCR] variable regions) (T Cell Diagnostics, Cambridge, MA). For CD3
depletion OKT3 (American Type Culture Collection [ATCC], Manassas,
VA) was used. Monoclonal antibodies were labeled with fluorescein
isothiocyanate, phycoerythrin, or allophycocyanin or were conjugated to
biotin. For flow cytometry, biotinylated antibodies were revealed by
streptavidin-phycoerythrin (BDIS), and unlabeled mAbs used were
revealed by GAM fluorescein isothiocyanate (BDIS). Isotypic control
antibodies were immunoglobulin G1 (IgG1) (X40 from BDIS) and IgG2a (X39
from BDIS). Anti-Fc
) or radiotherapy and
injection with TM-
). Each bar represents the mean ± SD
of 28 mice (RT) or 39 mice (RT + TM-
3.6 × 106), the number of TRECs would be
undetectable (less than 0.002 per 100 cells) if peripheral expansion of
contaminating injected T cells was solely responsible for the
peripheral T cells. Detection of significant levels of TRECs indicates
that in most peripheral CD45RA+ cells, T cells are recent
thymic emigrants and probably generate CD45RO+ cells on
activation.
