International Immunology, Vol. 14, No. 10, pp. 1113-1124,
October 2002
© 2002 Japanese Society for Immunology
The in vivo development of human T cells from CD34+ cells in the murine thymic environment
1 Department of Immunology, 2 Research Center for Genetic Engineering and Cell Transplantation, 3 Department of Surgery, and 4 Department of Hematology and Rheumatology, Tokai University School of Medicine, Kanagawa 259-1193, Japan 5 Central Institute for Experimental Animals, Kanagawa 259-1193, Japan
Correspondence to: S. Habu, Bohseidai, Isehara, Kanagawa 259-1193, Japan. E-mail: sonoko{at}is.icc.u-tokai.ac.jp
Transmitting editor: K. Okumura
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Abstract |
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There is increasing evidence that human hematopoietic stem cells can develop into lymphocytes expressing T cell surface markers in the organ culture of murine embryonic thymic lobes. If human T cells with functional maturity are inducible from human stem cells in the mouse, it may be a useful model to investigate human T cell development and the human immune response in vivo. To approach this, we produced a hybrid cluster of murine fetal thymic epithelial cells and human cord blood-derived CD34+ cells (hu/m cluster) using reaggregate thymic organ culture, and subsequently implanted it under the kidney capsule of NOD/SCID mice. The implanted hu/m cluster grew in volume under the kidney capsule and contained increased numbers of CD4+CD8+cells as well as CD4 or CD8 single-positive cells with low CD1a expression. These lymphocytes were also shown to possess activity for producing IL-2 and IL-4. Characteristics similar to human T cells also developed in the thymus of newly established mice lacking NK activity from NOD/SCID mice. These results indicate that functionally mature T cells can develop in vivo from human hematopoietic progenitors in the murine environment composed of thymic epithelial cells.
Keywords: human, T lymphocyte, thymus, transplantation
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Introduction |
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The development of human lymphocytes with immunological functions has been investigated using human lympho-hematopoietic stem cells (HSC) (1–3). An increasing number of reports have indicated that human HSC of any origin can develop into myeloid, erythroid and CD19+ pre-B cells in vitro as well as in vivo (3–7). However, T cell development may be an exception because T cell lineages ordinarily develop in a thymic microenvironment (8). More than 10 years ago, McCune et al. reported that human T cells develop in immunodeficient mice when human fetal thymus and fetal lymph nodes are implanted side-by-side under the kidney capsule and fetal liver cells (9). Later, there were several reports showing that human T cells develop from CD34+ cells isolated from human fetal liver or bone marrow in the transplanted human fetal thymus in SCID mice (10–13). This ‘human T cell-bearing mouse’ indicated that human HSC possess the differentiation potential for T cell lineages in mice if a certain appropriate thymic microenvironment is prepared. This system seemed useful not only for examining human T cell differentiation from stem cells, but it also appeared promising for investigating therapeutic reagents against pathogens for human T cells such as HIV. However, its widespread application may be limited because the fetal human thymus and/or liver are almost unavailable for ethical reasons.
Fisher et al. first provided a great hint for overcoming this ethical problem by demonstrating that CD4–CD8– [double-negative (DN)] human thymocytes can develop into CD4+CD8– [CD4 single-positive (SP)] and CD4–CD8+ (CD8SP) cells in the organ culture of murine fetal thymus lobes [human/murine (hu/m) hybrid fetal thymic organ culture (FTOC)] (14). Plum et al. reported that purified human CD34+ cells differentiate to mature T cells in hu/m hybrid FTOC (15). These reports encouraged us to investigate human T cell development and to generate mature human T cells using the xeno-thymic microenvironment. Consequently, there are increasing reports showing that human hematopoietic stem cells from bone marrow or cord blood develop into mature type T cells such as CD4SP and CD8SP cells through CD4+CD8+ [double-positive (DP)] cells in the murine thymic microenvironment (15–18). However, it still remains to be clarified whether the murine thymic environment is able to substantially substitute for the whole developmental process of the human thymus. For instance, it has not been conclusively reported whether human T cells develop in a hu/m hybrid FTOC to possess functional potential similar to those of the genuine human thymocytes, although down-regulation of CD1a, one of the maturation markers, is detectable in a portion of CD4SP cells (17).
To determine the functions of human T cells developed in the murine thymic environment, a relatively large amount of T cells is required. Since the efficiency of re-populating human cells in the thymic lobes is low in the hu/m hybrid FTOC (15), only a limited number of developed T cells is available for functional analysis. This shortcoming may be overcome by reaggregate FTOC (RTOC) (19), as hematopoietic stem cells can stereo-closely interact with murine thymic epithelial cells without the re-populating process. Based on these various reports concerning human T cell development in murine thymic lobes, it should be possible to establish systems in which the in vivo generation of human T cells is induced from hematopoietic stem cells in mice without using human fetal organs.
In this study, we showed two systems for generating human T cells in vivo. First, a short RTOC was performed with both murine fetal thymic epithelial cells and human cord blood CD34+ cells to stereo-closely interact mutually, and subsequently these hu/m hybrid clusters were implanted under the kidney capsule of NOD/SCID mice. In the implanted clusters, the developed human T cells showed pronounced cell growth and functional maturity with cytokine-producing ability in which murine thymic epithelial cells formed thymus-like tissues with a three-dimensional structure. Second, human CD34+ cells were i.v. injected into newly established NOD/SCID/
cnull mice, which are NOD/SCID lacking NK activity. These injected human cells migrated into the thymus of all the recipients and developed into functionally mature T cells.
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Methods |
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Antibodies
Anti-human mAb used were CD1a–FITC (B-B5; Diaclone, www.diaclone.com), CD3–FITC (Leu-4; Becton Dickinson, www.bd.com/ B-B11; Diaclone), CD4–allophycocyanin (Leu-3a; Becton Dickinson), CD8–phycoerythrin (PE) (Leu-2a; Becton Dickinson) and TCR
ß–FITC (WT-31; Becton Dickinson). Anti-mouse thymic stroma cells used were anti-mouse thymic pan-epithelium (clone MTS5; PharMingen, San Diego, CA) and anti-mouse thymic medullary epithelium (clone MTS10; PharMingen). Anti-mouse mAb [CD4–PE (L3T4), CD8–biotin (Ly-2) and TCRß–FITC (H57-597)] were purchased from PharMingen.
CD34+ cord blood cells
Umbilical cord blood was obtained from full-term, normal newborns and used according to the institutional guidelines. Mononuclear cells were separated by Ficoll-Hypaque density gradient centrifugation (d = 1.077; Amersham Pharmacia, www.apbiotech.com) and CD34+ cells were isolated on the MACS CD34 immunomagnetic isolation system (Miltenyi Biotec, Gladbach, Germany) as recommended by the manufacturer. After twice purification by MACS, collected cells were checked for purity, which was >98%. For the transplantation into NOG mice, CD34+ cells were stained with PE-labeled anti-CD4 and FITC-labeled anti-CD34, and CD34 SP cells were sorted by a FACS Vantage (Becton Dickinson). The purity of CD34+ cells was >99.99%.
RTOC in vitro system
RTOC was carried out using the protocol illustrated in Fig. 1. Fetal thymic lobes of BALB/c mice were isolated at day 15 of gestation and cultured in AIM-V medium containing 1.35 mM dGuO as described previously (20). dGuO-treated lobes were harvested, washed in AIM-V medium twice and incubated in 3 ml of 0.25% trypsin/0.02% EDTA in Ca/Mg-free PBS at 37°C for 30–40 min. Adding an equal volume of medium stopped the reaction and cells were suspended by vigorous pipetting. Debris was allowed to settle, and the dispersed stromal cells were removed in the supernatant, spun down and resuspended in 400 µl. Freshly isolated CD34+ cells were mixed with dispersed stromal cells at a ratio of 5:1–10:1 and spun into a pellet. After removal of the supernatant, the cell pellet was dispersed into slurry by careful mixing and then drawn into a fine capillary pipette. An aliquot of the slurry was expelled as a discrete standing drop on the surface of a nucleopore filter floating on the medium as used for conventional organ culture. These standing drops reformed thymic lobes within 12 h. Reaggregated lobes were harvested after 2–8 weeks. We termed this method the in vitro RTOC culture system, as described in Fig. 1.
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Implantation of lobes into NOD/SCID mice (RTOC in vivo system)
NOD/SCID mice were anesthetized with 2.5% Avertin (0.25 ml/body), and the left and right kidneys were sequentially exteriorized, a 0.5-mm incision was made in each kidney capsule, and two or three pieces of reaggregated lobes were implanted underneath the bilateral kidney capsule using fine forceps under a microscope. Over 95% of the mice were successfully implanted. At 6–14 weeks after implantation, grafts were cut out and used for further analyses. We termed this method the RTOC in vivo culture system, as described above.
Transplantation of CD34+ cells into NOG mice
We recently established NOD/Shi-scid/scid with c defect, NOD/SCID/cnull (SCID/NOD/cnull) (NOG) mice, which were made from NOD/SCID mice by backcrossing with IL-2Rc-deficient mice. NOG mice at 8–10 weeks old were maintained at the Central Institute for Experimental Animals (Kawasaki, Japan). CD34+ cells (5–7 x 105) were transplanted into sublethally irradiated (3.0 Gy) NOG mice i.v. At 6–12 weeks after implantation, thymi were cut out and used for further analyses.
Flow cytometry
Thymocytes harvested from RTOC were pelletted by centrifugation and resuspended in PBS containing 1% BSA and 0.1% NaN3. Subsequently, cells were stained with the panel of antibodies described above and analyzed in a FACScalibur (Becton Dickinson) flow cytometer. Lymphocyte gates were set on the basis of forward and side scatter profiles to correspond to the gate-set to control human lymphocytes (from healthy adult volunteers). Cut-off values for the quadrants were set after compensation for FITC versus PE versus Per-CP and allophycocyanin emission, based on the analysis of single, double, triple and quadruple staining of positive and negative control samples, and of the appropriate mouse IgG isotype controls.
ELISA
Thymocytes were cultured in 96-well round-bottom plates with 250 µl/well of AIM-V medium containing both phorbol 12-myristate 13-acetate (PMA) and ionomycin at final concentrations of 2.5 and 250 ng/well at 37°C for 24 h. Culture supernatants were collected, and IL-2 and IL-4 production was assayed by human IL ELISA kit (Endogen, www. endogen.com) as recommended by the manufacturer.
Histochemistry
Thymic lobes were fixed in 10% formalin solution, embedded in paraffin, sectioned (4 µm) and stained with H & E. For immunostaining, the lobes were freshly embedded in OCT compound (Miles, Elkhart, IL), cryostat sectioned (6 µm) and serially stained with saturating amounts of mAb (100-fold dilution) against thymic medullary epithelium or against thymic pan-epithelium (PharMingen) and horseradish peroxidase-conjugated anti-rat IgG (500-fold dilution).
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Results |
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Expression of T cell markers in cord blood CD34+-derived cells in hu/m hybrid cluster in RTOC
In murine FTOC, hematopoietic progenitor cells could not be efficiently seeded into thymic lobes, particularly in combination with human-derived cells (14,21). Instead of FTOC, we used RTOC in which most of the input cells interact directly with thymic epithelial cells without the seeding process (19). In RTOC, murine thymic epithelial cells isolated from 15-day fetuses were mixed with human cord blood-derived CD34+ cells by spinning and the slurry of mixed cell pellets was cultured on nucleopore filters to form the hu/m hybrid cluster. CD34+ cells isolated by the MACS CD34 immunomagnetic system (Miltenyi Biotec, Gladbach, Germany) showed >99% purity with <0.3% CD3+ cells.
In the first 2-week RTOC, human CD45+ cells in the hu/m hybrid cluster increased their cell number 
5-fold, but they were composed of >50% DN cells, 25% CD4SP cells, and small proportions of DP and CD8SP cells. Moreover, the CD4SP cells were mostly CD3–, indicating that they may have been mostly immature at the pre-DP stage, as indicated by Plum et al. (16). In the following RTOC analyses, the proportion of DP cells rapidly increased and CD4SP cells with high CD3 expression increased. The proportion of low CD1a and CD4SP cells, which are reported to be mature thymocytes (17), seemed to increase a little at a time, but the total cell number of the RTOC had markedly decreased. Consequently, the proportion of DP cells was decreased (Fig. 2 and Table 1A). These results indicated that human T cells that developed in the murine thymic environment expressed mature T cell markers, but their cell numbers are not sufficient for detecting T cell functions such as cytokine production.
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Implantation of hu/m hybrid clusters provides marked cell growth in thymic lobular structures
To determine whether lymphoid cells and epithelial cells are retained intact in hu/m hybrid clusters on nucleopore filters of long RTOC, the clusters were morphologically examined. Tissue sections of clusters in the first 4-week RTOC showed diffuse distribution of many lymphoid cells among spindle cells with clear abundant cytoplasm and large nucleus (Fig. 3A-a). However, the hu/m hybrid clusters in 6-week RTOC contained large fatty droplets, sparsely distributed lymphoid cells, and spindle cells that were strongly and homogeneously stained with eosin (Fig. 3A-b). These histological features indicate that thymic epithelial cells in hu/m hybrid clusters become degenerated on nucleopore filters during longer culture, which may result in the lack of supporting ability for lymphoid cell survival in RTOC.
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To retain the three-dimensional structure composed of intact thymic epithelial cells, hu/m hybrid clusters were implanted under the kidney capsule of NOD/SCID mice after 1-week RTOC. The implanted clusters became progressively larger over 6–8 weeks as shown in Fig. 3(B). The cluster tissues showed histological features of the thymic lobular structure, in which numerous lymphoid cells were found in the mesh of spindle cells. The spindle cells were stained through most tissues with anti-cortical epithelium antibody and partially with anti-thymic medullary epithelium antibody (Fig. 3B-a–f). These results suggest that the implantation of hu/m hybrid clusters into NOD/SCID mice may contribute to the intact thymic epithelial cells and the generation of the thymic three-dimensional structures, resulting in the proliferation of lymphoid cells in a much better condition than in in vitro culture.
In vivo development and proliferation of human mature type T cells in implanted hu/m clusters
Lymphocytes grown in implanted hu/m clusters in NOD/SCID mice were mostly human CD45+ cells (data not shown) and greatly increased in cell number, ranging from 94- to 200-fold (Table 1B) during 6–12 weeks of implantation. To determine whether these in vivo proliferating CD45+ cells were T cells at the developing stages, flow-cytometric analysis was performed. The expression profiles of CD4/8 for these CD45+ cells showed that they consisted of >90% DP cells, and small proportions of CD4SP (5%) and CD8SP (2%) cells from week 6 to 10 (Fig. 4 and Table 1B). The majority of CD4SP and CD8SP cells expressed high levels of CD3 and TCRß molecules (>80%, see Fig. 4), with higher expression levels than DP cells (data not shown). These findings indicate that both CD4SP and CD8SP cells in the implanted hu/m hybrid clusters were mostly post-selected mature T cells. At the same time, the proportion of low CD1a CD4SP cells gradually increased up to 50% during 6–12 weeks after the implantation. These phenotypic profiles of T cells seemed to be similar to those of in situ human thymocytes.
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From these results, it seems clear that human CD34+ cells can develop into mature T cells with cell growth through physiological processes in hu/m hybrid clusters implanted in NOD/SCID mice as effectively as in the human thymus. At the moment, we do not know which molecules in thymic epithelial cells are essential for T cell development, but at least we know that they may be shared between humans and mice.
Cytokine-producing potential is induced in human T cells from implanted hu/m clusters
According to surface molecules that mark T cell maturity, human T cells seem to be functionally developed in the implanted hu/m clusters with marked cell proliferation, as seen in the in vivo thymus. Then, we examined whether or not these human T cells in the implanted clusters substantially acquire T cell functional potentials as peripheral T cells do.
For this purpose, cell suspensions were obtained from hu/m clusters during 6- to 12-week implants, and analyzed for the production potentials of IL-2 and IL-4 in vitro. After in vitro stimulation of these thymocytes with PMA plus ionomycin, ELISA measured the amounts of IL-2 and IL-4 in culture supernatants. In comparison with non-stimulated cells, IL-2 production was greatly enhanced, 5-fold, in the stimulated human T cells of 6-week implants and increased up to 10-fold in 8- to 12-week implants along with the increased proportion of low CD1a CD4SP cells. In contrast to IL-2, IL-4 production was relatively low, but still significantly increased in the stimulated cells. Such cytokine production was almost undetectable in human lymphoid cells obtained from in vitro RTOC (data not shown).
These results indicate that in vivo developed human T cells expressing mature-type surface molecules actually possess cytokine-producing potential for IL-2 and IL-4 if the murine thymic epithelial cells are retained intact and generate three-dimensional structures.
Mature T cells are detected in the thymus of NOD/SCID/cnull mice
The above results from the implanted hu/m clusters indicate that human CD34+ cells can efficiently proliferate and develop into functionally mature T cells in vivo if human hematopoietic progenitors or stem cells migrate into the murine thymus. To confirm this issue, we used recently established NOD/SCID/c null mice, termed NOG mice (submitted), because they completely lack NK cell activity, whereas it is slightly detectable in NOD/SCID mice (22). Sixteen NOG mice were used for thymic analysis after being injected with human cord blood-derived CD34+ cells (2 x 105). The CD34+ cells were carefully purified by a FACS Vantage and the contamination of CD3+ cells was <0.01%. All the NOG thymi we examined (100%, 10 heads) contained various numbers of CD45+ cells ranging from 2 to 75 x 105 during 6–12 weeks after CD34+ cell injection (Fig. 6). In contrast, such a high frequency of human cells was never found in the thymi of NOD/SCID mice after receiving human cord blood CD34+ cells (28%, eight of 28 heads). Moreover, the CD45+ cell number in the thymus was much smaller in NOD/SCID mice, ranging from 1 to 5 x 104, than in NOG mice.
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Flow-cytometric profiles of human CD45+ cells in the NOG thymus showed that the proportions of subpopulations divided by CD4 and CD8 expression are similar to those observed in the implanted hu/m hybrid clusters after 8 and 10 weeks of CD34+ cell injection (Fig. 6). The DP cell proportion was
80%, accompanied by 10% of CD4SP and 8% of CD8SP at weeks 8 and 10, although the CD8SP cell proportion was low at week 6. More than 50% of CD4SP cells and CD8SP cells expressed a high level of TCRß/CD3 and a low level of CD1a, suggesting that these cells were post-selected T cells with functional maturity. In fact, cytokine production of IL-2 and IL-4 was also detectable after in vitro culture with PMA plus ionomycin for 24 h (Fig. 7) in human T cells developed in the NOG thymus 12 weeks after CD34+ cell injection.
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CD3+ cells were also detectable in the peripheral blood and spleen in these mice (3–30% of total human CD45+ cells), and were almost CD4 and CD8SP cells (data not shown). These CD3+ cells showed marked proliferation when they were cultured in the presence of anti-CD3 antibody (Table 2). These human T cells are considered to develop from the CD34+ cells at least for the following reasons. When we transplanted 106 purified CD34+ cells (99.99%) in one NOG mouse, only 100 T cells (corresponding to 0.01% of 106) in total were estimated to be distributed through the NOG body if these contaminated cells were all mature T cells. These may be out of the range of detection by FACS analysis. In fact, when NOG micereceived 100 human mature T cells isolated from the peripheral blood i.v., these T cells were not detectable over several months by FACS (data not shown).
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Discussion |
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For the in vivo development of human T cells with functional maturity, we produced a hybrid cluster of murine fetal thymic epithelial cells and human cord blood CD34+ cells using RTOC, and subsequently implanted it under the kidney capsule of NOD/SCID mouse. In the implanted hybrid cluster, human CD34+ cells remarkably increased and developed into mature-type T cells with CD1alow expression. These human T cells developed in vivo by stereo-closely interacting with murine thymic epithelial cells were functionally mature, and possessed the potential for producing cytokines such as IL-2 and IL-4. The in vivo generation of mature human T cells was also prominent when cord blood-derived CD34+ cells were injected into NOG mice, which completely lack NK activity in addition to having the features of NOD/SCID mice.
Since the first surprising report by Fisher et al. (14), the hu/m hybrid FTOC system has been invaluable for the in vitro generation of human T cells from progenitor cells at various stages (14–16,18,21,24), because human fetal tissues such as the thymus and liver are not needed. However, most of the studies did not investigate the T cell function of developed human cells in hu/m hybrid FTOC. Among those, Res et al. reported that the down-regulation of CD1a is a marker of the functional maturity of CD4-expressing human thymocytes (17) in FTOC. Consequently, later studies reported that the low expression of CD1a was observed in a portion of CD4SP cells (18), or in CD5+ cells or CD3+ cells (24), which were developed from human CD34+ cells in murine FTOC. However, it has not been clearly shown which T cell functions are acquired in human T cells that developed in the murine thymic environment.
To address this issue, a relatively large cell number is required. However, the seeding of human progenitors into the murine thymic lobe represents at least 20% of the input cells in FTOC [data not shown and (21)]. Thus, we employed RTOC instead of FTOC. In the hu/m hybrid RTOC, most of the applied CD34+ cells have a chance to easily contact with the thymic epithelial environment, since the dissociated murine thymic epithelial cells are forced to directly interact with any human progenitor cells without any entry process into the thymic lobes. Moreover, the applied cells may not be selected for exclusive seeding of certain particular progenitor cells in the xeno-combination as indicated by previous reports (14,21). In hu/m RTOC, human CD34+ cells substantially develop into thymocyte subpopulations such as DP and CD4SP and CD8SP cells during the first 4–6 weeks. However, the three-dimensional structure of thymic epithelial cells composed by RTOC seemed to be lost during the long culture that was required for inducing a number of mature-type T cells (Fig. 3), resulting in a marked decrease of human-derived lymphoid cells in RTOC.
Implantation of the hu/m hybrid cluster made by short RTOC under the kidney capsule of NOD/SCID mice contributed to the retention of the three-dimensional structure of thymic epithelial cells and to the promotion of marked cell growth. The implanted hybrid clusters showed very similar histological features to the freshly isolated thymic lobes containing numerous lymphoid cells among the distinct cortex and medulla. The intrathymic cells were almost human CD45+ cells, and were composed of good proportions of DP cells and SP cells with low CD1a expression. It is also notable that the proportion of CD4SP versus CD8SP in the implanted cluster was nearly equal, with a similar profile to that of freshly obtained human thymocytes. On the other hand, the proportion of CD4SP cells was always dominant in RTOC alone (Fig. 3) and in FTOC seen in the previous reports (15,18,23,24). Using these human T cells, we first demonstrated that human T cells developed in hu/m hybrid clusters have the ability to produce cytokines such as IL-2 and IL-4.
In addition to the advantages such as marked cell growth and functional maturation of human T cells, this in vivo modified RTOC may be useful for manipulating not only human progenitor cells but also thymic epithelial cells because both are divided in a short RTOC (data not shown).
Having established that human cord blood-derived CD34+ cells functionally developed by stereo-close interaction with murine thymic epithelial cells, human CD34+ cells are presumed to develop into T cells in mice if they migrate into the thymus. In comparison to the murine/murine combination, human stem cells migrated into the thymi of NOD/SCID mice at a low frequency, as shown in both of our present study and in another report (6). However, if our recently established NOG mice are used as recipients, injected human CD34+ cells and/or their progenitors migrate into the thymus of almost all mice to develop mature T cells (Fig. 6). There are several reports showing that engraftment of human hematopoietic stem cells (22,25) or human CD4+ T cells (26) is enhanced in NOD/SCID mice lacking NK cell activity by administration of anti-acialo-GM1 or anti-IL-2Rß. Moreover, ß2-microglobulin-deficient NOD/SCID mice, which also lack NK cell activity, enhanced the human T cell engraftment (27). Thus, we predict that higher engraftment of implanted human cells might result in the increase of human cells developing in the thymus of NOG mice without NK cell activity.
As clearly shown in our present study, the in vivo development of functional human T cells from hematopoietic progenitors is successful in the microenvironment of murine thymic epithelial cells in two experimental systems. These systems are useful for both investigating the mechanism of human T cell development, and inspecting the T cell sensitivity to reagents and pathogens or T cell development after gene manipulation. In addition, we found relative proportions of CD3+CD4+ or CD3+CD8+ human T cells in the periphery in NOG mice. As discussed in Results, they are considered not to be contaminated human T cells in cord blood cells. However, it is not evident at the moment whether they are descendants developed in the thymus or extrathymically developed T cells (28). Based on the surface markers, these cells in the periphery may not be NK cells because they were almost all CD16–CD56– cells (data not shown). This point is needs clarification and is now under investigation.
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Acknowledgements |
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The authors thank Daisuke Suzuki, Tatsuya Okada, Masato Yoshioka, Yasuyuki Hirano and Ikumi Katano for technical assistance, and the Makino Clinic and the members of the Tokai CBSC Study Group for providing human cord blood. This study was supported by The Japan Society for the Promotion of Science (JSPS) grant no. JSPS-RFTF97100201; a Grant-In-Aid for Scientific Research and a Research Grant of The Science Frontier Program from the Ministry of Education, Science, Sports and Culture of Japan; a Grant-In-Aid from the Ministry of Health and Wealth; and grants from 1998 Tokai University of Medicine Research Aid and the Japan Research Foundation for Clinical Pharmacology.
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Abbreviations |
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DN—double negative
DP—double positive
FTOC—fetal thymic organ culture
HSC—lympho-hematopoietic stem cell
hu/m—human/murine hybrid
PE—phycoerythrin
PMA—phorbol 12-myristate 13-acetate
RTOC—reaggregate fetal thymic organ culture
SP—double positive
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