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a Immunology Department, Weizmann Institute of Science, Rehovot, Israel;
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4 P! X d; z' B) t/ \# \- cb Etablissement Francais du Sang Bourgogne/Franche-Comte, Besancon, France;# L! Z0 V9 Y# \; O9 |
; K* V' K2 u; wc Gene Therapy Institute, Hadassah University Hospital, Jerusalem, Israel;% j# c' u3 Z/ n; N7 ~% [
- y9 ~# F! q" C- ud Hematology and Bone Marrow Transplantation Department, Chaim Sheba Medical Center, Tel-Hashomer, Israel;
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; K, J, g: |3 ?# }5 |8 Ae Department of Obstetrics and Gynecology, Assaf-Harofeh Medical Center, Zerifin, Israel;# K& U' `7 v; B" T! k$ {
6 ^5 `# k, ?, Z: B+ Q9 Jf Institut de Genetique Moleculaire de Montpellier, Montpellier, France- ^" c e3 l* X& A5 r9 {, G4 P7 T
6 Q; A( W# @( K) W' e& D i; ~Key Words. TNF ? Transplantation ? T lymphopoiesis ? Cord blood ? Mobilized peripheral blood cells ? Hematopoietic stem cells! h( _9 j: c p& w- @
# ?& Q$ k' H0 y0 J* ZCorrespondence: 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@weizmann.ac.il
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ABSTRACT
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/ {* \+ L- A- F$ h/ _1 L$ pT-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 . This process continues throughout adult life, although at a reduced rate . 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 . Understanding the mechanisms of human T-cell development and function is thus important to enhance immune reconstitution after clinical HSC transplantation.
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7 Z2 b, u! s; g1 O7 L1 uTo better elucidate these processes, a variety of experimental systems for studying human T lymphopoiesis have been used . 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 . This approach enables studies to be performed within the thymic tissue, but development of mature CD8 thymocytes is rare , 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) . 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 . A defect in migration of human T-cell progenitors to the mouse thymus was suggested by van der Loo et al. , 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.
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) j/ {+ k0 I$ X) N+ VOther studies using in vivo models include the SCID-hu model, in which human CD34 cells are coimplanted with T cell–depleted human fetal thymus . 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 . 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.0 x$ Y) K% {8 w; G# E, s
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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 or by knockout of the IL-2Rg gene . 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.6 l ]" E: @( E5 w- z9 I8 i) X4 y7 B
0 J8 K: Q1 Y6 V) K# CExtrathymic differentiation of T cells plays an important role in immune reconstitution in the absence of efficient thymic function. Strober and colleagues have reported the presence of early murine T-cell progenitors in the adult mouse BM that generate CD4 and CD8 T-cell receptor (TCR)-? 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 and after transplantation of human CB CD34 progenitor cells transduced with an active form of Notch into NOD/SCID recipient mice .9 E' v# _0 i! f* d O) [
8 j" g+ d5 ?" O8 M) {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 . Weekx et al. 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 .
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) w: _: X4 H: V6 sThe 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).6 N- W4 P1 w* N
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MATERIALS AND METHODS
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6 V, k+ n+ \& d, |- s0 a9 ~. Y" s' t; TTNF 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* r. w# c- O7 K: @, @$ Z
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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 CD45high 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 CD45high 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. y4 B, U+ `2 Q
& a1 \8 U& q$ e0 h; r7 @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.
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; K6 N8 X, j# D/ _" a, E" _ TIn preliminary experiments performed to determine the optimal protocol for promoting T-cell repopulation, we injected TNF at different time points before, during, and after transplantation of total MNCs, CD3 -depleted MNCs, or CD34 -enriched cells (data not shown). Analyses of the different protocols examined demonstrated that human T-cell engraftment was obtained only when total MNCs were transplanted together with TNF administration 4 hours before transplantation. Therefore, we established this procedure as the standard protocol for our subsequent transplantation experiments. TNF-treated recipient mice rarely showed any signs of graft-versus-host disease (GVHD) within 2 months after transplantation.2 R" y6 @# A* k7 }: Z a" q8 E2 t
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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).% _' k% H1 P _
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TNF Treatment In Vivo Leads to the Emergence of DP Pre-T Cells 2–3 Weeks after Transplantation
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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 (
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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.1 m# t' A- `7 H5 d) \: C- h7 E, P* h
3 j6 J% |- ]1 }9 w. j, h7 W9 o0 \0 XEngrafted Human T Cells Are Derived from Immature CD34 -Enriched Cells
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To additionally substantiate that the reconstituted T cells originated from immature CD34 progenitors, we performed sex-mismatch studies using Y chromosome as a marker. Enriched CB CD34 cells from male donors were separated by two columns and determined to be devoid of mature CD3 T cells (Fig. 3A). These cells were then cotransplanted into NOD/SCID mice together with CB CD34– MNCs from a female donor. One month after transplantation, the CD4/8 DP and SP subsets recovered from the BM of the recipients were separated by antibody-magnetic bead column and used for PCR amplification of Y-chromosome DNA-specific sequences. Figure 3B shows that Y-chromosome sequences are present in the separated human T cells, as well as in the human T-negative separated cells. Such approach shows that the engrafted human T cells were mostly of male origin, indicating that they derived from the immature CD34 progenitor cells. Of note, transplantation of 20 million CD34– MNCs (which include approximately 4 million mature T cells) or transplantation of purified, 4 million mature T cells did not engraft or, in rare cases, led to only negligible levels of human cell engraftment only by MNC, most probably because of low CD34 cell contamination. In addition, enriched CD34 cells transplanted alone did not give rise to T cells within 1 month after transplantation (data not shown).
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) K. _3 A4 D7 z; qFigure 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.
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TNF-Pretreatment In Vivo Enhances Human T-Cell Reconstitution from G-CSF–Induced MPBL Transplanted Cells5 t% Z% H8 D+ D( v
) `1 M4 j1 E" y3 w0 ?" f3 _1 U0 BWe next examined whether TNF also enhances T-cell engraftment in hematopoietic repopulating cells derived from sources other than CB. We thus chose to study adult G-CSF–induced MPBLs, because they are the major source of HSCs for clinical transplantations. The experimental protocol of TNF pretreatment was identical to that used for CB cells. The results show that TNF enhanced both total and T-lineage engraftment from human MPBL (total engraftment, 4.4 ± 1.6% in TNF-treated mice versus 1.7 ± 1% in mice without TNF treatment, Fig. 4A), but the levels of engraftment were in general lower than those detected in CB MNC-transplanted mice. The high variability in levels of engraftment was probably attributable to variability in SRC frequency and expression of CXCR4, a receptor critically involved in homing and engraftment, in different adult donors in general . However, the trend was similar in all experiments. As shown in Figure 4B, human MPBL transplanted into TNF-treated mice gave rise to human T cell engraftment, whereas myeloid and B cells were rarely found. The frequency of human T-cell engraftment, determined by the presence of greater than 0.5% DP cells in the murine BM, was 43% (6 of 14) in TNF-pretreated mice versus 21% (3 of 14) in control mice. It thus appears that TNF-treatment has a beneficial effect on human T-cell reconstitution after MPBL as well as CB transplantation in NOD/SCID mice." e& F4 ] r3 J' B5 r0 z8 z
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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., v0 H7 B( i& F% T
- j7 w. P0 G% J* t) x& S. G }Human T Lymphocytes Reconstitute the Hematopoietic Organs and the Peripheral Circulation of TNF-Treated NOD/SCID Recipients/ R# {1 k, E$ a# j1 a6 a* x1 S
/ ]* j# V) F" x& K8 H$ fTo assess whether reconstitution of human T cells in NOD/ SCID mice after TNF treatment leads to T-cell repopulation in lymphoid and hematopoietic tissues other than BM, we studied the engraftment of human T lymphocytes in the spleen, thymus, and peripheral blood (PB) of recipient mice 1 month after transplantation of either CB MNC or MPBL (Fig. 5). As shown in Figure 5A, human T- cell repopulation was not restricted to the BM; human T cells were also detected in spleen and PB of TNF-treated recipient mice. Most important, we detected immature human T cells also in the thymus of these mice, the major organ for T-cell development. Levels of T-cell engraftment were generally similar in all of the organs examined (Fig. 5B), although engraftment in the murine thymus appeared at lower levels. Human cell engraftment levels were higher in mice transplanted with CB MNCs compared with those transplanted with MPBL in all organs examined. In mice that were not treated with TNF and did not show human T cells in their BM, human T cells could not be detected in their spleen, thymus, or PB (data not shown).
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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.& l v% {9 J* f) G& E; H4 o
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Phenotypic Characterization of the Engrafted Human T Lymphocytes in TNF-Treated NOD/SCID Mice- f* f+ n: i0 s( c
9 [' Q0 A7 p- B& ZTo further characterize the nature of the T cells engrafted in TNF-pretreated recipient mice, T lymphocytes from the BM and thymus were analyzed for additional markers of T cells. The phenotype of human T lymphocytes from a representative experiment is shown in Figure 6. Most of the T cells expressed CD3 on their surface. Mature SP CD4 CD3 and CD8 CD3 T cells were rapidly detectable within 3 weeks after transplantation. Further phenotypic analysis showed that the human CD3 cells expressed TCR?, and no substantial levels of TCRcells were detected. Importantly, expression of CD1a, a marker of immature thymocytes that disappears on terminal differentiation, was detected only in the thymus at low levels (Fig. 6, thymus). Phenotypic analyses for naive (CD45RA ) and memory (CD45RO ) cells in the BM demonstrated an abundance of CD45RO memory cells when CB MNCs were transplanted. In the case of MPBL transplantation, all of the engrafted human T cells were CD45RO (data not shown). Table 1 summarizes the results of the relative percentages of SP and DP T-cell populations, calculated in relation to the total T-cell engraftment levels. When CB MNCs were transplanted, the levels of CD8 SP and CD4 SP T cells in the murine BM were mostly similar, although there was variability from mouse to mouse. When MPBLs were transplanted, the levels of CD8 SP cells were higher than CD4 SP cell levels (2:1 ratio).
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& B5 I- y: d0 `' _2 V" ?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.
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Table 1. Proportion of human CD4 and CD8 single- and double-positive T cells in tumor necrosis factor–treated nonobese diabetic/severe combined immunodeficiency recipient mice
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To examine the clonality of human T cells engrafted in TNF-pretreated NOD/SCID mice, hypervariable complementarity determining region 3 size distribution of 24 variable regions of ? chain (V?) of the TCR subsets was analyzed by the immunoscope method. Analysis of TCR-V? expression exhibited a wide range of TCR-V? usage in the BM and spleen of some TNF-pretreated recipient mice. Results of T cells from the BM of one recipient mouse are shown in Figure 7. The results were variable with greater and reduced diversity in individual mice. Nevertheless, the data demonstrate the reconstitution of human T cells expressing multiple TCR-V? chains.0 p! t* |# \2 k. g! E! Z
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Figure 7. CDR3 size distribution of human TCR-V? subsets of T cells developed in TNF-treated mice. The TCR-V? 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-V? subfamily-specific primers and one constant consensus primer (C?), followed by a run-off reaction with a fluorescent C? primer. The graphs represent fluorescence intensity in arbitrary units plotted against the amino acid size of CDR3. The size distributions within nine TCR-V? families from BM of one recipient mouse are shown. A polyclonal profile is observed for TCR-V? 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.
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; T3 l/ v1 w5 M1 ^" J; \( |5 i5 v) [We sought to evaluate development of T cells by analysis of TRECs, which are formed during the TCR rearrangement process that T cells undergo during maturation in the thymus, in additional experiments with seven human donors. Because recently differentiated T cells contain a certain TREC content, quantification of this TREC content is considered a measure of T lymphopoiesis. DNA was isolated from human T cells separated from the BM and spleen of recipient mice 1 month after transplant and analyzed for TREC content by quantitative PCR (numbers of human T cells detected in the murine thymus in this stage were not sufficient to perform TREC analysis). TRECs were detected in T cells isolated from most samples, including spleen as well as BM (Table 2). Nevertheless, levels of TRECs were approximately 5 to 60 per million cells, which is relatively very low, suggesting that these cells have undergone multiple rounds of division after differentiation, as previously observed in both murine and human transplantation studies in which T cells differentiated from HSCs . Alternatively, we cannot rule out that these low levels are also attributable to proliferation of the transplanted mature T cells.+ Z5 G! F; p0 O4 O" J3 [
: Z/ n% q$ l5 z9 Y; z+ Q+ {8 iTable 2. TREC content of human CD4 and CD8 cells recovered from tumor necrosis factor–treated recipient mice1 D8 _1 \4 |6 t) W I
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DISCUSSION
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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.& Y" c/ {* H1 v7 W$ @
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