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Tumor Necrosis Factor Promotes Human T-Cell Development in Nonobese Diabetic/Severe Combined Immunodeficient Mice

^a Immunology Department, Weizmann Institute of Science, Rehovot, Israel;
^b Etablissement Francais du Sang Bourgogne/Franche-Comte, Besancon, France;
^c Gene Therapy Institute, Hadassah University Hospital, Jerusalem, Israel;
^d Hematology and Bone Marrow Transplantation Department, Chaim Sheba Medical Center, Tel-Hashomer, Israel;
^e Department of Obstetrics and Gynecology, Assaf-Harofeh Medical Center, Zerifin, Israel;
^f Institut de Genetique Moleculaire de Montpellier, Montpellier, France

Key Words. TNF • Transplantation • T lymphopoiesis • Cord blood • Mobilized peripheral blood cells • Hematopoietic stem cells

Correspondence: Tsvee Lapidot, Ph.D.,Weizmann Institute of Science, Department of Immunology, PO Box 26, Rehovot, 76100, Israel. Telephone: 972-8-9342481; Fax: 972-8-9344141; e-mail; Tsvee.Lapidot{at}weizmann.ac.il

	ABSTRACT

Top Abstract Introduction Materials and Methods Results Discussion References

 
A major problem after clinical hematopoietic stem cell transplantations is poor T-cell reconstitution. Studying the mechanisms underlying this concern is hampered, because experimental transplantation of human stem and progenitor cells into nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice usually results in low T–lymphocyte reconstitution. Because tumor necrosis factor (TNF) has been proposed to play a role in T-lineage commitment and differentiation in vitro, we investigated its potential to augment human T-cell development in vivo. Administration of TNF to irradiated NOD/SCID mice before transplantation of human mononuclear cells from either cord blood or adult G-CSF–mobilized peripheral blood (MPBL) led 2–3 weeks after transplantation to the emergence of human immature CD4⁺CD8⁺ double-positive T-cells in the bone marrow (BM), spleen, and thymus, and in this organ, the human cells also express CD1a marker. One to 2 weeks later, single-positive CD4⁺ and CD8⁺ cells expressing heterogenous T-cell receptor ß were detected in all three organs. These cells were also capable of migrating through the blood circulation. Interestingly, human T-cell development in these mice was associated with a significant reduction in immature lymphoid human CD19⁺ B cells and natural killer progenitors in the murine BM. The human T cells were mostly derived from the transplanted immature CD34⁺ cells. This study demonstrates the potential of TNF to rapidly augment human T lymphopoiesis in vivo and also provides clinically relevant evidence for this process with adult MPBL progenitors.

	INTRODUCTION

Top Abstract Introduction Materials and Methods Results Discussion References

 
T-cell progenitors develop from hematopoietic stem cells (HSCs) that migrate from the bone marrow (BM) to the thymus, where they undergo a sequential program of proliferation, differentiation, and selection to generate mature functional T-cells for export to the peripheral blood and tissues [1]. This process continues throughout adult life, although at a reduced rate [2]. Limitation of this process is a major cause for early immune dysfunction in adult patients undergoing HSC transplantation. Depressed T-cell development in these patients has been related mainly to low levels of thymic function in adults [3,4]. Understanding the mechanisms of human T-cell development and function is thus important to enhance immune reconstitution after clinical HSC transplantation.

To better elucidate these processes, a variety of experimental systems for studying human T lymphopoiesis have been used [5]. One of the in vitro strategies is the fetal thymus organ culture (FTOC), in which primitive human progenitors are seeded onto lymphoid-depleted murine fetal thymic lobes [6,7]. This approach enables studies to be performed within the thymic tissue, but development of mature CD8⁺ thymocytes is rare [8], and differentiation is uncoupled from the in vivo–occurring migration and homing of cells. Severe combined immunodeficient (SCID) and nonobese diabetic (NOD)/SCID mice have been used as a functional preclinical model for in vivo engraftment of human hematopoietic progenitor cells. This system identified a small population of primitive hematopoietic cells capable of repopulating transplanted recipients, defined as SCID-repopulating cells (SRCs) [9–13]. However, using these models, studies of T-cell reconstitution after HSC transplantation are limited because of the low and slow development of human T cells [11,14]. A defect in migration of human T-cell progenitors to the mouse thymus was suggested by van der Loo et al. [15], who succeeded in inducing homing of human cells to the thymus of NOD/SCID mice previously transplanted with enriched human CD34⁺ cells by G-CSF and stem cell factor (SCF)–induced mobilization.

Other studies using in vivo models include the SCID-hu model, in which human CD34⁺ cells are coimplanted with T cell–depleted human fetal thymus [16]. Hence, both FTOC and SCID-hu models are based on the availability of a fetal thymic tissue. In another model, extrathymic T-cell development can be achieved in the athymic immunodeficient bnx mouse by cotransplantation of human BM CD34⁺ stem and progenitor cells together with human BM stromal cells engineered to produce human interleukin (IL)-3 and IL-7 [17]. However, significant numbers of human T cells can be recovered from the BM of these mice only 4–6 months after transplantation, and there is no human B-cell development in these mice.

Models that were recently developed are based on the inhibition of natural killer (NK) cell activity in NOD/SCID and Rag2 knockout mice either by neutralizing antibodies against the murine IL-2R [18] or by knockout of the IL-2Rg gene [19,20]. In these models, human T cells develop in the murine thymus, and mature peripheral T cells begin to emerge 8–12 weeks after transplantation of enriched human cord blood (CB) CD34⁺ cells.

Extrathymic differentiation of T cells plays an important role in immune reconstitution in the absence of efficient thymic function. Strober and colleagues [21,22] have reported the presence of early murine T-cell progenitors in the adult mouse BM that generate CD4⁺ and CD8⁺ T-cell receptor (TCR)-ß⁺ T cells in an extrathymic pathway, supported by the marrow microenvironment. Similarly, extrathymic differentiation of human T cells in mouse BM was documented both in the bnx/hu model [17] and after transplantation of human CB CD34⁺ progenitor cells transduced with an active form of Notch into NOD/SCID recipient mice [23].

The cytokine tumor necrosis factor (TNF), produced by a variety of cell types, including thymic stromal cells, was found to be associated with critical events leading to T-lineage commitment and differentiation. In mice, TNF induces CD25 (IL-2R) expression on early immature thymocytes and is required for additional thymocyte maturation to single-positive (SP) CD4 and CD8 cells [24]. Weekx et al. [25] showed that TNF also promotes differentiation of human BM CD34⁺-enriched cell populations into T cells in the FTOC system. However, T-cell differentiation and selection, as well as the thymus architecture, are normal in TNF knockout mice. Interestingly, there is a threefold increase in B-cell numbers in the thymus of these mice [26].

The present study was designed to determine whether TNF can augment human T-cell development in vivo. We established a system that rapidly generated human T cells in vivo, based on TNF pretreatment to irradiated NOD/SCID mice before transplantation of either human CB mononuclear cells (MNCs) or, even more clinically relevant, adult mobilized peripheral blood (MPBL).

	MATERIALS AND METHODS

Top Abstract Introduction Materials and Methods Results Discussion References

 
Human Cell Preparation
Human CB was obtained from full-term deliveries, and granulocyte colony-stimulating factor (G-CSF)–MPBL was obtained from healthy donors for clinical transplantation. All human cell samples were obtained and used in accordance with the procedures approved by the Human Experimentation and Ethics Committees of the Weizmann Institute. MNCs were isolated from samples by standard separation on Ficol-Hypaque (Pharmacia Biotech, Uppsala, Sweden) density gradient centrifugation and washed in phosphate-buffered saline. Enrichment of CD34⁺ cells for CD34⁺ cell transplantation experiments was performed using a MACS cell isolation kit and AutoMacs magnetic cell sorter (Miltenyi Biotech, Bergisch Gladbach, Germany) according to the manufacturer’s instructions, resulting in a purity of 98 ± 1%. No significant levels of CD3⁺ cells were detected by flow cytometric analysis (0.25 ± 0.15% CD3⁺ cells).

Human T-cell populations (CD4⁺CD3⁺, CD8⁺CD3⁺, and CD4⁺CD8⁺ subsets) were isolated from recipient mouse BM or from CB MNCs using magnetic beads directly conjugated to anti-CD3 and anti-CD8 Abs (Miltenyi Biotech) to exclude CD4⁺CD3^– myeloid cells.

Mice and Transplantation
NOD/LtSz-Prkdc^SCID (NOD/SCID) mice were bred and maintained under defined flora conditions at the Weizmann Institute in sterile microisolator cages. All of the experiments were approved by the Animal Care Committee of the Weizmann Institute. Mice (6–8 weeks old) were irradiated at a sublethal dose (375 cGy) from a cobalt source before transplantation. The following day, mice were injected i.p. with 0.5 µg human TNF (R&D Systems, Minneapolis). Four hours later, 20 x 10⁶ human CB MNCs or MPBLs were injected i.v. For CD34⁺ cell transplantation experiments, 2 x 10⁵ enriched CD34⁺ cells were injected with or without 20 x 10⁶ CD34^– MNC fraction. Mice were euthanized at various time intervals after transplantation; BM, spleen, thymus, and peripheral blood cells were harvested and resuspended into single-cell suspensions.

Flow Cytometry Analysis
Human hematopoietic cell engraftment was examined by flow cytometry analysis on FACSCalibur (Becton, Dickinson, San Jose, CA) using mouse anti-human CD45-fluores-cein isothiocyanate (FITC) monoclonal antibody (mAb) (Immuno Quality Products [IQP], Groningen, The Netherlands). Lineage cell analysis was performed by staining with anti-CD3-Cy-Q, CD45RO Phycoerythrin (PE), CD45RA-FITC (all from IQP), CD1a-PE, CD19-PE, CD56-PE, and anti-CD4-RD1/CD8-FITC (Coulter, Miami), TCRß-FITC (BD Pharmingen, San Diego), and TCR-PE (BD Pharmingen). Human plasma and mouse immunoglobulin G (IgG) were used to block human and murine Fc-receptors. Isotype-matched control antibodies were IgG1 FITC/IgG1 PE (IQP), used to exclude false-positive cells. Viable cells were gated by exclusion of propidium iodide–positive cells.

Polymerase Chain Reaction of Y Chromosome–Specific DNA
To identify T cells of male CB CD34⁺-donor origin in the recipient murine BM, sorted human progenitors and mature T cells were lysed in 10 mM Tris-HCl, pH 8.1, 400 mM NaCl, 10 mM EDTA, 1% SDS, and 750 µg/ml Proteinase K (Sigma, St. Louis) for 2 hours at 56°C. After inactivation of Proteinase K by saturated NaCl (6 M) for 5 minutes on ice, the samples were washed and DNA was extracted by ethanol. DNA from each sample (0.2 µg) was used for polymerase chain reaction (PCR) amplification of human Y-chromosome sequences. The Y chromosome–specific primers were as follows: forward 5'-TGGGCTGGAATGGAAAGGAA TCGAAAC-3' and reverse 5'-TCCATTCGATTCCATTTT TTTCGAGAA-3' [27]. PCR was performed for 28 cycles as follows: denaturation at 94°C for 1 minute, annealing at 62°C for 1 minute, extension at 72°C for 1 minute, and a 10-minute final extension at 72°C. PCR products were separated by electrophoresis on a 1.6% agarose gel and visualized by ethidium bromide.

TCR-Vß Repertoire Analysis
RNA (2 µg) was reverse transcribed with random hexanucleotides (Pharmacia Biotec, Orsay, France) using moloney murine leukemia virus reverse transcriptase (Gibco BRL, Cergy, France). cDNA was amplified (40 cycles) in a 25-µl reaction mixture with one of the 24 TCR-Vß subfamily–specific primers and a Cß primer recognizing the two constant regions Cß1 and Cß2 of the ß chain of the TCR, as previously described [28]. Two microliters of the 24 TCR-Vß/Cß first-run PCR product were subjected to two cycles of elongation (run-off) using a Cß dye-labeled (6-Fam) primer allowing PCR products to be detected on an automated DNA sequencer (Applied Biosystems, Foster City, CA). Run-off PCR products were loaded on a 6% acrylamide sequencing gel and analyzed for size and fluorescence intensity using the Immunoscope software. The TCR-Vß nomenclature proposed by Arden et al. [29] was used in this study.

T-Cell Receptor Excision Circle Quantification
The signal-joint T-cell receptor excision circles (TRECs) in separated human CD4 and CD8 T cells from recipient mice were quantified by means of real-time quantitative PCR with Taqman assays. The PCR primer sequences were as follows: sense 5'-GCCAGCTGCAGGGTTTAGG-3', antisense 5'-CATCCCTTTCAACCATGCTGACACCTCT-3'. The probe sequence was as follows: 5'-FAM-ACACCTCTGGTTTTT GTAAAGGTGCCCACT-TAMRA-3'. DNA was extracted from the murine BM and spleen cells as described above for Y-chromosome PCR. Each PCR reaction was performed from 500 ng of genomic DNA with the use of Master Mix PCR probe x 2 Quantitect^TM (Qiagen, Courtaboeuf, France) containing Hotstar Taq DNA polymerase, deoxynucleotide triphosphate mix, and 8 mM MgCl₂, 10 pmol of each primer, and 5 pmol of fluorescent probe. One cycle of denaturation/ enzyme activation (95°C for 10 minutes) followed by 50 cycles of amplification (94°C for 30 seconds, 60°C for 30 seconds) was performed on LightCycler (Roche Diagnostics, Meylan, France). A series of standard dilutions of a plasmid containing the signal-joint breakpoint was used to quantitate TRECs in each mouse and control DNA sample. Cycle threshold was assessed using the second derivative method with the LightCycler software 3.5.3. Each DNA sample was run in duplicate. Quantification of a reference gene (GAPDH) was done with the same conditions with primers and probe as follows: GAPDH1: 5'-GAGATGGTGCAGAACCTCAT-3', primer GAPDH2: 5'-CCAAATTCATCGAAATAGCC-3', and GAPDH probe: 5'-6FAM-CACCACAGAGGCCCAAG GTC-TAMRA-3'. Results are expressed as mean of duplicate TREC copies number normalized by GAPDH gene copy number.

Statistical Analysis
Results are expressed as mean ± standard error of the mean values. Significance levels of difference between experimental groups were determined by Student’s t-test.

	RESULTS

Top Abstract Introduction Materials and Methods Results Discussion References

 
TNF Promotes Reconstitution of Human T Lymphocytes from CB SRC with a Concomitant Reduction in Human Immature B Lymphocytes in the BM of NOD/SCID Recipient Mice
The potential involvement of TNF in human T-cell development was studied by injection of TNF into sublethally irradiated NOD/SCID mice and transplantation of human CB MNCs 4 hours later. The activity of human TNF on the mice was confirmed by examination of adhesion molecule expression in the murine BM. We found that human TNF upregulated inter cellular adhesion molecule (ICAM)-1 expression (twofold increase) on murine BM cells in vivo within 8 hours (data not shown). One month after transplantation, BM cells of recipient mice were analyzed for expression of the human CD45 marker to evaluate total human cell engraftment in TNF-treated mice. Levels of total human cell engraftment in TNF-treated mice were similar to those detected in untreated mice (50 ± 9.7% and 47.4 ± 7.9%, respectively; n = 8). However, flow cytometric analyses showed the emergence of a CD45^high cell population in the BM of TNF-treated mice, which was not apparent in the BM of untreated mice, parallel to a reduction in immature CD19⁺ B cells (Fig. 1A). We additionally analyzed the CD45^high cells by staining with CD4, CD8, and CD3 markers of human T cells and found that these cells were T lymphocytes (Fig. 1B; CD3 staining is not shown). Some of these cells were CD4⁺CD8⁺ double-positive (DP), phenotype characteristic to immature T cells, strongly suggesting that TNF treatment induced human T-cell development. The frequency of human T-cell engraftment, determined by the presence of more than 0.5% DP cells in the murine BM, was 46% (27 of 59) in TNF-pre-treated recipient mice versus 25% (11 of 43) in control recipient mice.

View larger version (32K): [in this window] [in a new window]   Figure 1. Human cell engraftment in the BM of CB MNC-transplanted NOD/SCID. NOD/SCID mice were sublethally irradiated (375 cGy) and were either pretreated or not (control) with 0.5 µg TNFper mouse injected i.p. Four hours later, mice were transplanted with 20 x 106 human CB MNC per mouse (i.v.). BM cells were collected from individual recipient mice 1 month after transplantation, stained with human-specific monoclonal antibodies, and analyzed by flow cytometry for the presence of human cells. Gates were set on side-scatter (SSC) versus forward light scatter (FSC) and on viable cells. (A): Representative analysis of human cells in an individual recipient mouse as assessed by the percentages of CD45+ human hematopoietic cells and CD19+CD45+ B cells. (B): Presence of human T cells in representative BM sample as assessed by monitoring CD4 and CD8 expression. (C): Percentage of human lymphocytes engrafted in murine BM. Each dot represents one mouse, and bars indicate the mean value. (D): Cells recovered from the murine BM were differentiated in vitro with human stem cell factor plus interleukin-15 (100 ng/ml each) for 10 days and then stained for the expression of the CD56 natural killer cell marker. Percentages of human cells are indicated. *Significant difference in T- and B-cell engraftment between TNF-pretreated mice and untreated mice (p = .01). Abbreviations: BM, bone marrow; CB MNC, cord blood mononuclear cell; NOD/SCID, nonobese diabetic/severe combined immunodeficient; TNF, tumor necrosis factor.

T-cell engraftment after TNF treatment led to a concomitant reduction in the levels of immature human B cells (Fig. 1A). As demonstrated in Figure 1C, the percentages of developing human B cells in the BM were 31 ± 5.5% (n = 7) in control mice compared with 12.6 ± 3.9 (n = 7) in TNF-treated mice (p = .01). The ability of human lymphoid progenitors from the BM of TNF-treated mice to differentiate ex vivo into CD45⁺CD56⁺ NK cells in response to IL-15 and SCF was also lower than that of progenitors from control untreated mice (Fig. 1D).

TNF Treatment In Vivo Leads to the Emergence of DP Pre-T Cells 2–3 Weeks after Transplantation
To determine whether the human T cells detected in mouse BM 1 month after transplantation were derived from the expansion of transplanted mature T cells that homed to the BM or, alternatively, whether they developed in the mouse, we followed their kinetics in the BM of the recipient mice. A low percentage of human T cells that homed to the BM was detected 24 hours after transplantation (<3.5%, data not shown), but the cells were not detectable after 1 week (Fig. 2). Two weeks after transplantation, CD4⁺ cells were detected at a slightly higher level in the BM of TNF-treated compared with untreated mice (10.6 ± 2.3% versus 7.3 ± 1.2%; n = 4). In the TNF-treated mice, there was also a stepwise emergence of a small population of DP pre-T cells and SP CD8⁺ cells (<0.4%). These populations increased at 3 and 4 weeks after transplantation in BM of TNF-treated mice but were not detected during the entire length of the experiment in mice that were not treated with TNF. Thus, these kinetic studies, especially the emergence of CD4⁺ pre-T cells followed by DP cells 4 weeks after transplantation, together with the reduction in human B and NK lymphocyte progenitors, suggest in vivo development of T cells in response to TNF within a relatively short period of time after transplantation.

View larger version (35K): [in this window] [in a new window]   Figure 2. Kinetics of human T-cell development in the BM of transplanted mice. Irradiated nonobese diabetic/severe combined immunodeficient mice were either pretreated or not (control) with TNF before transplantation of 20 x 106 cord blood mononuclear cells. BM cells were collected from individual recipient mice at various time points, and the presence of human T cells was monitored by staining with human-specific CD4/CD8 surface-marker monoclonal antibody. Representative flow cytometric analyses are shown, and the percentages of single-positive and double-positive T cells are indicated. Each plot represents different mouse. Abbreviations: BM, bone marrow; TNF, tumor necrosis factor; w, week.

View larger version (40K): [in this window] [in a new window]   Figure 3. Detection of CD34+-derived T cells in the BM of NOD/SCID recipient mice. Irradiated NOD/SCID mice pre-treated with TNF were transplanted with 2 x 105 human cord blood male CD34+-enriched cells together with 20 x 106 female CD34– MNCs. (A): Representative flow cytometric analysis of the purity of male CD34+-separated cells. The purified cells are devoid of CD3+ mature T cells. (B): One month after transplantation, the human CD4/CD8 double- and single-positive BM subsets were separated. DNA was extracted and assessed for the presence of Y chromosome–specific sequences by polymerase chain reaction. The resulting products resolved on a 1.6% agarose gel are shown. A band with the predicted size of 150 bp is detected in the positively selected BM T cells as well as in the remaining T-negative cells, indicating the colonization of the BM by progenitors of male origin. As a positive control, DNA from CD34+ cells of male donors is shown, and as a negative control, CD34– female MNC DNA is shown. The H2O lane shows a control using Y chromosome–specific primers and no template DNA. Abbreviations: BM, bone marrow; MNC, mononuclear cell; NOD/SCID, nonobese diabetic/severe combined immunodeficient; TNF, tumor necrosis factor.

View larger version (23K): [in this window] [in a new window]   Figure 4. Human cell engraftment in the BM of MPBL-transplanted NOD/SCID mice. NOD/SCID mice were sublethally irradiated (375 cGy) and were either pretreated or not (control) with 0.5 µg TNF per mouse injected i.p. Four hours later, mice were transplanted with 20 x 106 human adult MPBL per mouse i.v., and BM cells were collected from individual recipient mice 1 month after transplantation. (A): Total human cell engraftment within BM of recipient mice determined by CD45 staining. Each dot represents one mouse, and bars indicate the mean value of 11 mice from three independent experiments. (B): Representative flow cytometric analysis of individual recipient mouse (percentages are indicated). Abbreviations: BM, bone marrow; MPBL, mobilized peripheral blood; NOD/SCID, nonobese diabetic/severe combined immunodeficient; TNF, tumor necrosis factor.

View larger version (28K): [in this window] [in a new window]   Figure 5. Human cell engraftment in murine organs of TNF-pretreated NOD/SCID mice. Cells were collected from individual recipient mice 1 month after transplantation of human CB MNC or MPBL, stained with human-specific mAbs, and analyzed for the presence of human cells. (A): Representative flow cytometric analysis of human cells in the BM, spleen, thymus, and PB of NOD/SCID mouse transplanted with CB MNC (percentages are indicated). (B): Percentage of human CD4+ and CD8+ single- and double-positive cells in the BM, spleen, and thymus of recipient mice transplanted with either CB MNC or MPBL. Each bar represents the mean ± standard error of the mean of at least seven mice transplanted with CB MNC and three mice transplanted with MPBL. *Significant difference in T-cell engraftment between CB and MPBL for BM (p = .05). Abbreviations: BM, bone marrow; CB MNC, cord blood mononuclear cell; MPBL, mobilized peripheral blood; NOD/SCID, nonobese diabetic/severe combined immunodeficient; PB, peripheral blood; TNF, tumor necrosis factor.

View larger version (31K): [in this window] [in a new window]   Figure 6. Phenotypical analysis of human T cells engrafted in CB nonobese diabetic/severe combined immunodeficient recipient mice. BM and thymus cells from tumor necrosis factor–pretreated mice transplanted with CB mononuclear cells were collected 1 month after transplantation, stained with human-specific monoclonal antibody, and analyzed by flow cytometry for different markers of human T cells. Representative results are presented (percentages are indicated). Abbreviations: BM, bone marrow; CB, cord blood; TCR, T-cell receptor.

View larger version (28K): [in this window] [in a new window]   Figure 7. CDR3 size distribution of human TCR-Vß subsets of T cells developed in TNF-treated mice. The TCR-Vß repertoire was assessed by analysis of TCR CDR3 size distribution (Immunoscope profiles) of BM and spleen cells obtained from TNF-pretreated mice 4 weeks after transplantation of human mononuclear cells. Twenty-four PCR products were generated by RT-PCR with 24 different TCR-Vß subfamily-specific primers and one constant consensus primer (Cß), followed by a run-off reaction with a fluorescent Cß primer. The graphs represent fluorescence intensity in arbitrary units plotted against the amino acid size of CDR3. The size distributions within nine TCR-Vß families from BM of one recipient mouse are shown. A polyclonal profile is observed for TCR-Vß families 3, 7, 8, 9, 13a, 14, and 18, and a skewed profile is observed for TCR-6b and 11. Abbreviations: BM, bone marrow; CDR3, complementarity determining region 3; RT-PCR, reverse transcription–polymerase chain reaction; TCR, T-cell receptor; TNF, tumor necrosis factor.

	DISCUSSION

Top Abstract Introduction Materials and Methods Results Discussion References

We report here that pretreatment with TNF resulted in a 1.9-fold increase in the success rate of human T-cell generation in NOD/SCID recipients within 1 month after transplant. This effect of TNF, which is produced also by thymic stromal cells, is in agreement with previous data on murine and human T lymphopoiesis. Zuniga-Pflucker et al. [24] have shown that TNF is important for the induction of critical events leading to murine T-lineage commitment and maturation in a thymus reconstitution assay, acting either directly on lymphocyte precursors or indirectly on a non–T cell subset that is critical for T-cell maturation. In line with these findings in mice, Weekx et al. [25] showed that, in the in vitro FTOC system, TNF promotes T cell differentiation of CD34⁺ cells from adult human BM, which we have also observed for human CB-derived CD34⁺ cells (our unpublished data). Of interest, genetic polymorphism leading to increased TNF production may enhance susceptibility to adult T-cell leukemia/lymphoma among human T-lymphotropic virus I carriers [34]. However, studies in TNF-deficient mice show no abnormalities in thymic stromal architecture and level of the cell yield, T-cell differentiation, positive selection, and function [26,35], suggesting a compensation for the lack of TNF by other factors. Interestingly, in support of our observation that immature human B-cell levels decreased in parallel with an increase in T-cell levels in TNF-treated mice, a threefold increase in B-cell level was observed in the thymus of TNF knockout mice [26]. Because of the rapid clearance of human TNF from the murine circulation [36], the effect of TNF seems to be indirect in the present study, because the transplanted human cells are injected 4 hours after TNF treatment. It is possible that TNF affects the stromal cells in the murine organs, which, in turn, affect the differentiation into T lineage of the human stem and progenitor cells, which are transplanted later. Examination of adhesion molecule expression in the murine BM in response to the human TNF used in this study confirmed its activity on the treated mice by demonstrating upregulation of ICAM-1 expression. Because murine TNF-R2 (p75) shows strong specificity for murine TNF, whereas murine TNF-R1 (p55) has similar affinity for both murine and human TNF [37], the activities of human TNF observed in our model are probably mediated by mTNF-R1, which is known to initiate signals for cytotoxicity and protective activity [38,39]. The finding that engraftment of B cells and NK progenitors was reduced whereas that of T cells increased is similar to that observed in previous studies on murine and human HSCs transduced with constitutively active Notch [23,40]. When these transduced cells are transplanted into mice, they differentiate toward T cells in the murine BM and exhibit a simultaneous absence of early B-cell lymphopoiesis. The effect of TNF may thus be mediated by factors directing common lymphoid progenitors to T-lineage commitment, resulting in reduced number of B and NK progenitors. However, further investigation is required to resolve the molecular consequences of TNF in this matter.

We show here that TNF enhanced T-cell reconstitution also from adult G-CSF–mobilized PBL, which is the primary source of hematopoietic stem and progenitor cells for autologous and allogeneic transplantations. Previous studies with human MPBL reported that transplantation of either CD34⁺ or MNC into NOD/SCID mice results in engraftment of erythrocytes, myeloid, and B cells with an absence of T lymphocytes [15, 41, 42]. Van der Loo and colleagues [15] found immature human T cells in the murine thymus only in recipient mice treated with the mobilizing factors G-CSF and SCF 2 months after transplantation of approximately 20 x 10⁶ MPBL-CD34⁺ cells. However, no human T cells were detected in BM, spleen, or PB of these mice. These observations suggested the existence of a barrier between species that prevents rapid repopulation of the irradiated mouse thymus by human progenitors, probably attributable to a defect in the recruitment and migration of human T-cell precursors from the murine BM to the murine thymus. In the present study, levels of human cell engraftment after MPBL transplantation were lower than in the case of CB transplantation, consistent with previous reports documenting lower frequencies of SRCs in G-CSF-MPBL compared with CB [41,43]. Yet TNF treatment of the recipient mice increased the human cell engraftment level and T-cell frequency, whereas B and myeloid cells were rarely detected. Furthermore, human T cells could be detected in the BM, spleen, PB, and, more important, the thymus of the recipient mice, indicating that they were capable of migration and dissemination through the circulation.

The possibility that the human T cells observed in the recipient mice represent solely an expansion of the initial mature T cells that were included in the transplanted MNCs is highly unlikely, because the sex-mismatch transplantation study demonstrates that the origin of at least a portion of the mature T cells in these mice is the progeny of transplanted immature CD34⁺ cells. Moreover, (a) no CD3⁺ cells could be detected in the enriched male CD34⁺ cells before their transplantation, as shown in Figure 3A; (b) sequential presence of CD4⁺CD3^– cells further develops into DP immature T cells, with expression of human CD1a in the murine thymus, a marker that is absent on mature human T cells before their transplantation; (c) heterogeneous TCR-Vß repertoire is found, in contrast to human T cells engrafted in hu-PBL/SCID mice, that initially start from a varied TCR-Vß repertoire followed by severe restriction over time to xenoreactive clones [44,45]; and (d) only rare signs of GVHD are observed. In addition, neither a transplantation of 20 million CD34^– MNCs nor transplantation of 4 million purified T cells resulted in engraftment of human cells 1 month after transplantation (data not shown). In support of our conclusion, it has been reported that circulating mature T cells were rarely detected in NOD/SCID mice without coimplantation of human thymic transplant when they were reconstituted with CB MNCs [11,27]. However, in the present model, cotransplantation of different mature hematopoietic cells, including T cells, was required to initiate T-lineage reconstitution and development, and it is possible that these mature T cells also marginally contribute to the pool of human T cells seen 1 month after transplant.

Expression of human CD1a, indicating T-cell development, was detected only in the murine thymus. However, DP T cells were also found outside the thymus, and the highest level of human T cells was observed in the murine BM. There are now many lines of evidence for extrathymic T-cell differentiation, demonstrating that a microenvironment supporting T-cell development is not unique to the thymus [46–50]. As reported in previous studies, stromal cells in the murine BM may provide necessary and sufficient signals for the maturation of T cells from T-cell precursors [17, 21–23]. Strober and colleagues [21,22] found in mice that this process is strongly inhibited by the presence of mature T cells. However, in the TNF-pretreated NOD/SCID recipients, we found that the presence of mature human T cells was necessary for the development of human T cells, because transplantation of CD3⁺-depleted MNCs did not give rise to T-cell engraftment. This observation is in line with our previous data on human T-cell development in the FTOC system, in which the appearance of human DP cells depended on the presence of either CD4⁺ or CD8⁺ mature T cells in the cultured CB samples [6]. Mature T cells may regulate the development of human T lymphocytes from HSCs by production of T cell–promoting cytokines in the murine microenvironment, at least in the early stage of engraftment. Such cytokines may act either directly on the immature cells or on the stromal microenvironment. Additionally, mature human B cells and myeloid cells can serve as antigen-presenting cells and therefore may be required for human T-cell education in the murine microenvironment. Alternatively, the phenotype of DP T cells may also represent population of mature activated T cells, because Kitchen et al. [54] reported that CD8 cells can re-express CD4 on their surface on costimulation. However, our kinetics studies demonstrate that CD4 cell appearance precedes CD8 and DP T-cell appearance and CD8 cells do not appear before DP cells and therefore are in favor of a T-cell differentiation process rather than activation.

In support of our results, Plum and colleagues [23] also reported the development of human CD4⁺CD8⁺CD3⁺ T lymphocytes in the BM of NOD/SCID recipient mice 2 months after transplantation of CD34⁺ cells transduced with an active form of Notch. CD19⁺ B lymphocytes were nearly absent within the transduced population. In the bnx/hu mouse model, human T cells are recovered from the BM of bnx mice in the greatest numbers 6 months after transplantation, suggesting a slow differentiation. In the present study, levels of DP T cells are relatively low compared with those levels observed in murine models, where human T lymphopoiesis takes place in the murine thymus—NOD/ SCIDc^null and Rag2^nullc^null mice [19,20] and blocked IL-2R NOD/SCID mice [18]—supporting the notion that BM T lymphopoiesis is much less efficient than thymic T lymphopoiesis. However, in the above models, human T cells began to emerge in the murine thymus only 4–8 weeks after transplantation. In contrast, we were able to detect human DP T cells in TNF-treated NOD/SCID mice, including low levels in the murine thymus, within 3 weeks, implying that the time required to detect human T cells in the present model seems to be shorter.

Because DP cells were also found in the BM and spleen, we added experiments to analyze their TREC content. TREC levels were detected in most samples of T cells isolated from the BM and spleen. Nevertheless, the relatively low level of TREC (5 to 60 per million cells) strongly suggests that these cells have undergone multiple rounds of division. This is likely attributable to the lymphopenic environment of these mice. Similar assessments have been made in previous transplantation studies in which T cells differentiated from HSCs, both in mice transplanted with T cell–depleted BM and in human T cell–depleted MPBL-transplanted patients (albeit of the same species). Indeed, the number of TRECs in proliferating T cells derived from HSCs has been reported to be approximately 10 per million cells [32,33], similarly to TREC levels observed in our study, derived from xenotransplantation. Accordingly, Hazenberg et al. [52] re-evaluated the use of TREC assay for measuring thymic output and concluded that TREC data should be interpreted with caution. Based on these reports, low TREC content in recipient mice does not necessarily provide evidence for suppressed T-cell production but may reflect dilution of TRECs resulting from increased proliferation after differentiation. However, we cannot rule out other possibilities, such as that these low levels reflect proliferation of the transplanted mature T cells or, alternatively, that only partial human T-cell development occurred in the murine BM and spleen.

Phenotypic analysis of the T cells that reconstituted TNF-pretreated recipients showed that, despite the variability, the overall CD4/CD8 ratio is 1:1 and 1:2 after CB and MPBL transplantation, respectively. Most of the T cells expressed CD45RO, suggesting that human T cells generated in the mouse might undergo stimulation by foreign antigen or, alternatively, undergo switching as a result of homeostatic proliferation. The cells exhibit a diverse TCR repertoire, and mice usually did not suffer from GVHD symptoms. Preliminary functional analyses of these engrafted T cells, based on their proliferative capacity in response to PHA or IL-2, indicate that they could indeed undergo activation, but to a much lesser extent than CB MNCs (data not shown).

Taken together, our study describes an animal model that can serve as a rapid and useful tool for studying human T-cell development, including from adult human MPBL in vivo, as well as factors that can enhance this process. Further research on the nature of this thymic and possible extrathymic T lymphopoiesis will allow us to evaluate the potential and contributions of these pathways to immune recovery after clinical HSC transplantations, especially in adult patients having low thymic function.

	ACKNOWLEDGMENTS

Top Abstract Introduction Materials and Methods Results Discussion References

 
This work was supported in part by a grant from AFIRST France-Israel grant agency. We would like to thank Professor Dov Zipori for fruitful discussions and to Loya Abel for helpful technical assistance.

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Top Abstract Introduction Materials and Methods Results Discussion References

 

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