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Keywords:

  • Committed T-lymphoid progenitors;
  • Human peripheral blood stem cells;
  • OP9/N-DLL1;
  • T-cell re-constitution;
  • Three-dimensional matrix

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results and discussion
  5. Concluding remarks
  6. Materials and methods
  7. Acknowledgements
  8. References
  9. Supporting Information

T-cell re-constitution after allogeneic stem cell transplantation (alloSCT) is often dampened by the slow differentiation of human peripheral blood CD34+ (huCD34+) hematopoietic stem cells (HSCs) into mature T cells. This process may be accelerated by the co-transfer of in vitro-pre-differentiated committed T/NK-lymphoid progenitors (CTLPs). Here, we analysed the developmental potential of huCD34+ HSCs compared with CTLPs from a third-party donor in a murine NOD-scid IL2Rγnull model of humanised chimeric haematopoiesis. CTLPs (CD34+linCD45RA+CD7+) could be generated in vitro within 10 days upon co-culture of huCD34+ or cord blood CD34+ (CB-CD34) HSCs on murine OP9/N-DLL-1 stroma cells but not in a novel 3-D cell-culture matrix with DLL-1low human stroma cells. In both in vitro systems, huCD34+ and CB-CD34+ HSCs did not give rise to mature T cells. Upon transfer into 6-wk-old immune-deficient mice, CTLPs alone did not engraft. However, transplantation of CTLPs together with huCD34+ HSCs resulted in rapid T-cell engraftment in spleen, bone marrow and thymus at day 28. Strikingly, at this early time point mature T cells originated exclusively from CTLPs, whereas descendants of huCD34+ HSCs still expressed a T-cell-precursor phenotype (CD7+CD5+CD1a+/−). This strategy to enhance early T-cell re-constitution with ex vivo-pre-differentiated T-lymphoid progenitors could bridge the gap until full T-cell recovery in severely immunocompromised patients after allogeneic stem cell transplantation.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results and discussion
  5. Concluding remarks
  6. Materials and methods
  7. Acknowledgements
  8. References
  9. Supporting Information

T-cell re-constitution critically influences outcome and treatment-related mortality after allogeneic stem cell transplantation (alloSCT). Normalization of the T-cell compartment after myeloablative therapy requires thymus-derived T-cell neogenesis; however, thymic resources are often compromised due to a damaged thymic microenvironment and older recipient age 1. Several strategies to accelerate thymic re-constitution identified in animal models have either not yet shown positive results in human trials (e.g. use of cytokines such as keratinocyte growth factor) or are associated with significant toxicity (e.g. androgen blockade). One interesting new approach is the co-transplantation of pre-differentiated lymphoid progenitors together with uncommitted HSCs. Committed lymphoid progenitors are present in vivo only at extremely low frequencies, but can be induced experimentally in the presence of Notch-ligand expressing (e.g. Delta-like-1 or -4) stroma cells 2, 3. Several phenotypes of committed T/NK-lymphoid progenitors (CTLPs) have been described 4, 5, all of which are strongly biased toward T-cell and NK-cell lineage development and exhibit an enhanced thymus-seeding capacity. Two recent publications have reported a rapid intrathymic engraftment of human CD34+CD45RA+CD7+ lymphoid progenitors after intrahepatic transplantation in neonatal mice 6, 7. However, in these two models, no extrathymic mature T cells could be detected, so it remained questionable whether a single intravenous injection of CTLPs can lead to peripheral T-cell engraftment.

The aim of our study was to analyse the developmental potential of in vitro-generated CTLPs transplanted together with haploidentical, G-CSF-mobilised CD34+ peripheral blood (huCD34+) HSCs in a murine model of humanised chimeric haematopoiesis. Our results show that CTLPs further differentiate after co-transplantation with huCD34+ HSCs in vivo, but not in vitro, and create an early wave of peripheral T-cell re-constitution at a time when progeny of huCD34+ HSCs is still at an early T-cell-progenitor stage.

Results and discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results and discussion
  5. Concluding remarks
  6. Materials and methods
  7. Acknowledgements
  8. References
  9. Supporting Information

HuCD34equation image HSCs differentiate into CTLPs on culture with murine OP-9/N-DLL-1 stroma cells

G-CSF-mobilised and purified huCD34+ HSCs were mainly lineageneg, CD34+38+, HLA-DR+CD117+, CD71+CD64 and CD45RACD7 (Fig. 1A and B). However, upon co-culture with OP9/N-DLL-1 stroma cells they rapidly acquired the described CD34+lineagenegCD45RAhighCD7+ phenotype (Fig. 1A, day 10) 4. Around 40% of cells acquired cytoCD3 and in part also CD5 by day 30 (Fig. 1C, upper plots); however, even after prolonged culture (until day 45 in two experiments), no expression of surCD3 (Fig. 1C, lower plots) or TCRαβ/γδ (data not shown) could be observed. About half of the CD7+ CTLPs expressed CD5 but only a minor fraction of these had already acquired CD1a (Supporting Information Figure 1A and B). As reported, CD4 increased after acquisition of CD5 or CD1a 6 but no CD4+CD8+ could be detected until the end of in vitro culture (Supporting Information Fig. 1B). To exclude that this maturation stop at the CD7+CD5+/−CD1a+/− level represents an intrinsic property of huCD34+ HSCs, we cultured CD34+-enriched cord blood progenitors (CB-CD34 HSCs) on OP9/N-DLL-1 stroma cells. Similar to their adult counterparts, CB-CD34 HSCs rapidly acquired the CD34+lineagenegCD45RAhighCD7+ phenotype but did not develop into mature CD3+ cells (Fig. 1B and C). Although two groups have reported the generation of mature single-positive T cells in OP9/DLL co-cultures 3, 8, others failed 7. Furthermore, questions remain about the functionality of these in vitro-matured T cells regarding their MHC-restriction and some phenotypic abnormalities 8. In accordance with our data, Meek et al. recently reported the same maturation arrest at the T/NK-progenitor cell level using a DLL-4 over-expressing stroma cell line 7. CD34+lineagenegCD10+CD24+ committed B-cell precursors were not generated in our OP9/N-DLL1 co-culture.

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Figure 1. Co-culture of huCD34+ HSCs on murine OP9/N-DLL1 stroma cells. (A) Phenotypic characterisation of G-CSF-mobilised and selected huCD34+ HSCs. Cells were gated on forward/sideward characteristics for lymphocytes/stem cells (left plot) and subsequently analysed for surface marker expression. (B) Co-expression of CD45RA and CD7 gated on CD34+lineage cells derived from huCD34+ (n=4, top) or CB-CD34 HSCs (n=1, bottom). CB-CD34 HSCs co-cultures were harvested on day 30. (C) Differential expression of cytoCD3/surCD3 versus CD5 on progenitors from the same experiments as (B) after 30 days of co-culture. Displayed cells were gated on SSClow/CD34+. (D) Colony-forming capacity for erythrocytes (CFU-E, white bars) or granulocytes/macrophages (CFU-GM, black bars) in freshly thawed huCD34+ HSCs versus CTLPs from huCD34+ or CB-CD34 HSCs was analysed after 14 days of culture in semi-solid medium. Data are presented as mean+SD from duplicate samples of the three donors; note break in the y-axis. (E) Microscopic images of a BFU-E and a CFU-M from CB-CD34 HSCs-derived CTLPs. Cells were cultured in semi-solid medium for 14 days and analysed directly without further staining by light microscopy using an inverted cell culture microscope (magnification: 40× objective lens, 10× ocular). Representative microscopic image of n=4 experiments.

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Colony-forming assays showed that freshly thawed huCD34+ HSCs preferentially formed colony-forming units of granulocytes/macrophages (CFU-GM) but also colony-forming units of erythrocytes (CFU-E) (Fig. 1D). CTLPs on day 15 had completely lost their CFU-E capacity but retained a minor CFU-GM-forming capability, resulting in more macrophage- than granulocyte-colonies (Fig. 1E). CTLPs from CB-CD34 HSCs maintained both CFU-E and -GM capacities; however, reduced by 90% compared with freshly thawed huCD34+ HSCs (Fig. 1D).

Pre-differentiated huCTLPs accelerate peripheral T-cell re-constitution in vivo

Next, we tested the in vivo-differentiation potential of CTLPs in the immunodeficient NOD-scid IL2Rγnull mouse model. After sub-lethal irradiation, these mice generally show a stable engraftment of huCD34+ HSCs after 10 wk in all haematopoietic lineages (including T cells), which is superior to that of conventional NOD-scid mice 9. We transplanted 6-wk-old animals intravenously with huCD34+ HSCs plus unsorted CTLPs from a haploidentical third-party donor. Control mice received only CTLPs, only huCD34+ HSCs, or no cellular support after irradiation. Ancestry of engrafting cells could be deduced to huCD34+ HSCs or CTLPs according to their expression of HLA-B07 (CTLPs were from a HLA-B07+, huCD34+ HSCs from a HLA-B07donor).

All mice survived until day 28, however, in the irradiation control and in mice receiving only CTLPs no engraftment of huCD45+ cells could be detected (Fig. 2A). This is in contrast with the previous reports in which CTLPs alone showed at least a thymic repopulating capacity 6, 7. However, in these experiments, CTLPs were given intrahepatically into neonatal mice, which is quite different to our experimental setting. Our design resembles more closely a possible clinical application and makes haematopoietic or lymphoid re-constitution solely driven by CTLPs unlikely. In contrast with this, recipients of huCD34+ HSCs and huCD34+ HSCs/CTLPs showed high levels of huCD45+ engraftment in spleen, BM and thymus (Fig. 2B). Interestingly, descendants from CTLPs could be found in the lymphoid as well as in the myeloid and monocytic compartment of the BM (Supporting Information Fig. 2B), reflecting the CFU data and current models of lineage plasticity in lymphoid progenitors 10. CTLPs further developed downstream the T-cell developmental pathway. In bichimeric mice, 42% of CD45+HLA-B7+ spleen cells were CD5+CD7+, compared with 15% in the CD45+HLA-B7 fraction and 5% in the spleen of the huCD34+ HSC controls (Fig. 2A). Similar percentages of cytoCD3+CD5+ cells could be found in the same gate (44 versus 22 versus 3%, data not shown). Most importantly, mature surCD3+ T cells appeared only in the HLA-B7+ fraction of mice with chimeric human haematopoiesis (14% of all HLA-B7+CD45+ spleen cells, Fig. 2A). Notably, these peripheral T cells were almost exclusively CD4+TCRαβ+. The reason for this CD4-dominance remains unexplained so far; however, huCD34+CD38 recently also showed an exclusive outgrowth of CD4+ T cells after in vitro culture on OP9/DLL1+-cells 11. These CD4+ T cells could have been selected on various MHC-class-II molecules as CD11c+HLA-DR+ cells could be detected from HLA-B7+ and from HLA-B7 backgrounds (Supporting Information Fig. 3C). Functional assays showed that after polyclonal stimulation these CTLP-derived T cells produced IFN-γ but not IL-4 (Fig. 2C). CDR3-size spectratyping showed BV-fragments in 7/26 analysed BV-families in chimeric mice, whereas in huCD34+ HSC controls faint bands could be detected in two BV-families (Fig. 2D).

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Figure 2. Engraftment of human CD45+ cells in NOD-scid IL2Rγnull mice. (A) Mice were sub-lethally irradiated with 300 cGy, transplanted with huCD34 HSCs, CTLPs, a combination of both or left without stem cell support and analysed after 28 days. Different phenotypes of spleen cells on day 28 post-transplant. As the irradiation control yielded completely identical results with the CTLPonly group, only results of the latter are shown. Gating strategy is detailed in Supporting Information Fig. 3. (B) Percentages of CD45+ cells in BM, spleen and thymus on day 28 post-transplant were determined by gating on SSClow/CD45+ (Supporting Information Fig. 2A). Data are presented as mean±SD of n=3 samples. (C) Splenic T cells detectable in chimeric mice were expanded for 10 days with IL-2 and IL-7, stimulated with PMA/ionomycin and analysed for their ability to produce IFN-γ or IL-4. Displayed cells were gated on CD45+/HLA-B7+/CD3+ as detailed in Supporting Information Fig. 3A. (D) Diversity of the TCR-repertoire in spleen was analysed by CDR3-size spectratyping. Five representative out of 26 analysed BV-families from one animal are shown (n=1).

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In our model, T-cell progenitors such as CD7+CD5+ as well as CD4+CD8+ descending both from CTLPs and from huCD34+ HSCs could be found in spleen (Fig. 2A), thymus (Supporting Information Fig. 3B) and BM (data not shown). However, CD7+CD5+CD1a+ early cortical T cells could be detected only in the HLA-B7 fractions, indicating that HLA-B7+ CTLPs had already differentiated beyond that checkpoint and lost their potential for long-term T-cell renewal (Fig. 2A). This observation was especially obvious in thymus, where almost no HLA-B7+ T-cell precursors were detectable on day 28 anymore, whereas in the HLA-B7 fraction CD7+CD5+CD1a+ cells dominated (Supporting Information Fig. 3B), which were all cytoCD3+surCD3 (data not shown).

Collectively, these data show that in vitro-pre-differentiated CTLPs have lost their capacity to engraft after intravenous transfer in an adult xenogenic environment, probably due to a lack of appropriate niches that foster homing, survival and differentiation of CTLPs. However, with support of undifferentiated huCD34+ HSCs, these CTLPs give rise to an early wave of de novo-generated, mature CD4+ T cells in the host and show some degree of lineage plasticity. Simultaneously, more sustained T-cell neogenesis from huCD34+ HSCs proceeds at a slower pace, resulting in mature, peripheral CD4+ and CD8+ T cells 8–10 wk after transplantation (9 and unpublished data). Most intriguingly, we found mature T cells differentiated from CTLPs not only in thymus but also in the periphery. This apparent discrepancy to the previous reports can be explained by substantial differences in the realisation of transplantation experiments: one group applied a one-log lower CTLP dose with a similar IL-7 supplementation 6, the other used equivalent numbers of CTLPs but no IL-7 7. However, the most important difference is that we co-transplanted CTLPs with huCD34+ HSCs whereas in the other studies, huCD34+ HSCs were used only as a separate control group. Therefore, we hypothesise that CTLPs and huCD34+ HSCs act synergistically in this setting as CTLPs alone failed to engraft and huCD34+ HSCs alone could not generate mature T cells after 4 wk. The combined use of these cell types seems to be a pre-requisite for full exploitation of the T-lymphoid regeneration capacity of our CTLPs. It will be interesting to investigate in further pre-clinical studies whether engraftment potential of CTLPs can be augmented by co-transfer of cell types without stem cell properties but the ability to interact with lymphoid progenitors such as certain DC subsets (TECK/CCL25) or keratinocytes (DLL4) 12.

huCD34+ HSC differentiation in an artificial thymic organoid

Finally, we tested whether T cells or at least CTLPs could be generated in a novel 3-D cell-culture system free of xenogenic stroma. This system has been reported to yield functional, single-positive T cells from huCD34+ HSCs after 14 days 13, 14. After 3 wk of co-culture, there was a significantly increased number of mononuclear cells in thymic but not in skin co-cultures (Fig. 3A and B). However, the majority of these cells appeared in the macrophage/immature monocyte region (Fig. 3A). Similarly, small numbers of CD3+ cells could be detected in cultures with or without huCD34+ HSCs, which disappeared when stroma cultures were pre-treated with fludarabine prior to initiation of co-culture (Fig. 3A and B). Clonality analysis showed a severely restricted TCR-repertoire with similar clonal expansions on days 14 and 21 of culture in some BV-families (data not shown), suggesting that the detected T cells in this system represent the progeny of expanded thymocytes and not de novo-generated T cells. In addition, huCD34+ HSCs rapidly lost their CD34 expression (Fig. 3C). No CD34+lineageCD45RA+, B or NK cells could be detected at the end of culture (data not shown).

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Figure 3. 3-D co-culture of huCD34+ HSCs on human thymic or skin stroma cells. (A) FSC/SSC plots after 3 wk co-culture of huCD34+ HSCs on thymic stroma cells (left). Negative controls included cultures without huCD34+ HSCs (middle) as well as cultures without huCD34+ HSCs but with a pre-treatment with fludarabine to deplete thymus-derived lymphocytes (right). (B) Absolute numbers of harvested cells per well after 2 (skin, black bars) or 3 (thymus, white bars) wk of co-culture are shown (left). Numbers of CD3+ T cells recovered at the end of co-culture with thymic or skin stroma cells are displayed (right). (C) Percentages of cells expressing CD34 in the lymphocyte/monocyte region in (A) on days 7, 14 and 21 of co-culture with thymus or day 14 with skin stroma cells are depicted. (B, C) Data are presented as mean+SD, number of experiments are indicated below the bars (B, C). Statistical analysis determined by the nonparametric Wilcoxon test for unpaired samples. (D) Gene expression of Delta-like-1, Delta-like-4 (left) and Notch-1 (right) in OP9/N-DLL-1 cells, human skin fibroblasts, freshly isolated human thymocytes and thymic epithelial cells was analysed by semi-quantitative real-time PCR. Results (mean of triplicate analysis) from n=2 samples were normalised against expression of haematopoietic cell kinase and compared with Ct-values of the respective molecules in BM. Note different scaling of the y-axes.

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One reason for the lack of T-cell differentiation in the 3-D matrix system could be inadequate DLL-1 expression on stroma cells, as signalling through DLL-1 or -4 has been demonstrated to be indispensable for T-cell development 2. In fact, comparative PCR-analysis showed that thymic epithelial cells expressed DLL-1 and -4 only slightly higher than the BM control, whereas our OP9/N-DLL-1 cells over-expressed DLL-1 more than 30-fold. As expected, gene expression of human DLL-4 could not be detected in the murine OP9 stroma cells (Fig. 3D). In contrast, Notch-1 was comparably expressed on all analysed cell types (Fig. 3D). Thus, a 3-D cell-culture matrix, although more closely mimicking thymic architecture, cannot compensate for an inadequate low expression of Notch-ligands on surrounding stroma cells.

Concluding remarks

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results and discussion
  5. Concluding remarks
  6. Materials and methods
  7. Acknowledgements
  8. References
  9. Supporting Information

Previous reports have already demonstrated the ability of CTLPs to create a temporally limited wave of intra-thymic T-cell engraftment 6, 7. We confirmed that in vitro-pre-differentiated CTLPs develop more rapidly into mature T cells in vivo than conventional huCD34+ HSCs. We could extend these results by showing substantial numbers of mature CD4+ T cells in the peripheral blood, thus indicating systemic T-cell engraftment derived from CTLPs even when these cells were generated from a third-party donor. This technique of CTLP-transfer together with conventional stem cell grafts offers several highly attractive advantages: (i) a short in vitro-culture time of 10–14 days reduces the risk of contamination or genetic instability, (ii) when co-transplanted with huCD34+ HSCs, these CTLPs are able to engraft in adult mice after intravenous transfer and (iii) CTLPs used for short-term T-cell re-constitution could potentially be generated and stored in larger quantities from haploidentical or even HLA-incompatible donors. Although several issues like CTLP-generation on non-xenogenic DLL+ stroma, engraftment kinetics, in vivo functionality of CTLP-derived T cells, and the impact of three different MHC backgrounds (host, donor 1, donor 2) on intra-thymic T-cell selection have to be addressed in further pre-clinical studies, our data strongly suggest that this strategy may present a promising tool for accelerating T-cell re-constitution.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results and discussion
  5. Concluding remarks
  6. Materials and methods
  7. Acknowledgements
  8. References
  9. Supporting Information

Haematopoietic progenitor and stroma cells

According to the institutional guidelines, backups of G-CSF mobilised and highly purified huCD34+ HSCs from patients who had succumbed to their underlying disease were allocated for research purposes before their final disposal. Human thymic tissue was provided by the Department of Cardiac Surgery from children who underwent correction surgery for inborn heart abnormalities, fragments of biopsied human skin by the Dermatology Hospital, and cord blood cells by the Department of Gynaecology, all University of Tübingen. The study was reviewed by the Ethics Committee of the University of Tübingen (Nr. ♯24/2003V).

OP9/N-DLL1 cell culture

HuCD34+ HSCs (7.5×104) were cultured on monolayers of murine OP9/N-DLL-1-over-expressing stroma cells (♯RCB2124, RIKEN Biosource Center, Japan) in the presence of IL-7 (5 ng/mL), Flt-3 (5 ng/mL) and SCF (10 ng/mL, Immunotools). Medium exchange and transfer on a fresh monolayer was carried out every 3–4 days. Cells were harvested at the indicated time points. For transfer experiments, CTLPs from day 15 were chosen because at this time point CD45RA/CD7 generally showed maximal expression on CD34+lineage cells.

Animals and transplantation

NOD.Cg-PrkdcscidIL2rgtmWjl/Sz mice (abbreviated as NOD-scid IL2Rγnull) were maintained under pathogen-free conditions as described previously 9. All animal procedures were reviewed by the animal care committee of the University of Tübingen (Nr. K1/07). Six-wk-old recipients were sub-lethally irradiated with 300cGy using a 137Cs irradiator (Gammacell 1000 Elite; MDS Nordion). Twenty-four hours later, 1.5×106 HLA-B7huCD34+ HSCs (n=3) with or without 8.5×106 15 days pre-differentiated HLA-B7+ CTLPs (n=3) were i.v.-injected into the tail vein of recipient mice. Control mice received 5×106 CTLPs or no cellular support after irradiation (n=2, each). T-cell engraftment was supported by weekly i.v. application of 20 μg of Fc-IL-7 fusion protein (kindly provided by Merck KgaA, Darmstadt, Germany). Re-constituted mice were kept under pathogen-free conditions and sacrificed after 4 wk. Thymus, spleen and BM were removed and analysed by flow cytometry, PCR and functional assays.

3-D cell-culture matrix

CellFoam cell-culture dishes 10 mm in diameter×1 mm in depth with an average pore density of 80 pores per inch (Cytomatrix) were pre-cultured with fragmented thymic or skin tissue as described previously 13. After 22–35 days purified huCD34+ HSCs (1×105) were added onto the stroma-pre-cultured CellFoam matrices. Medium was changed every 3–4 days and non-adherent cells were harvested on day 14 (skin) or 21 (thymus). In some of the control experiments, fludarabine (GRY-Pharma) was additionally used at a concentration of 4 μg/mL prior to huCD34+ HSCs seeding.

Quantitative real-time PCR

Expression levels of Notch-1 and its ligands DLL-1 and -4 were analysed using standard procedures on an ABI 7300 (Applied Biosystems, Darmstadt, Germany). Primer sequences can be obtained from the corresponding author upon request.

Flow cytometry

Supernatant cells from cell cultures or single-cell suspensions from spleen, thymus and BM of transplanted mice were analysed by flow cytometry (all CD markers obtained from BD) on a LSRII. Anti-HLA-B7 antibody was purchased from onelambda (BMT GmbH). The lineage cocktail, used to exclude committed haematopoietic precursors, contained CD3, CD14, CD15, CD19 and CD56 (all from BD).

CDR3-size fragment length analysis

TCR repertoire diversity was analysed using standard CDR3-size fragment size analysis. After RT-PCR, amplicons were detected on an ABI310 capillary sequencer and analysed with GeneMapper software (Applied Biosystems).

Colony-forming assays

Colony-forming capacity of stem cells was determined using a commercial CFC-assay (Stem Cell Technologies, containing SCF, GM-CSF, G-CSF, IL-3 and EPO). Briefly, 2×103 CD34+ or 2×104 CTLPs were cultured for 15 days in semi-solid medium and then analysed for the presence of colony-forming units of granulocytes/macrophages (CFU-GM) or erythrocytes (CFU/BFU-E) using an inverted cell-culture microscope (Leica Microsystems, DM IRB, Wetzlar, Germany).

Cytokine production

Splenocytes were expanded with 100 U/mL IL-2 und 5 ng/mL IL-7 (Immunotools) for 10 days and then stimulated with PMA (50 ng/mL) and Ionomycin (750 ng/mL, Sigma) with addition of BrefeldinA (10 μg/mL) for the last hour before analysis. Production of IFN-γ and IL-4 was measured by intracellular flow cytometry using standard procedures.

Statistical analysis

For statistical comparison of results, we used the nonparametric Wilcoxon test for unpaired samples. A p-value of <0.05 was considered statistically significant.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results and discussion
  5. Concluding remarks
  6. Materials and methods
  7. Acknowledgements
  8. References
  9. Supporting Information

The authors thank Dr. Gerd Klein, University of Tübingen for providing aliquots of cDNA from isolated thymic epithelial cells for PCR analysis. Furthermore, the authors thank Mohammed Alkahled for his dedicated animal care. The authors thank the Merck KgaA company (Darmstadt, Germany) for kindly providing aliquots of the Fc-IL-7 fusion protein. H. Z. is the recipient of a scholarship from the Jürgen-Manchot Foundation. This work was supported by a grant from the Wilhelm-Sander-Foundation (♯2003.023.1, awarded to M. E. and K. S.) and by the Tour of Hope Foundation (unrestricted research grant to the University Children's Hospital Würzburg).

Conflict of interest: The authors declare no financial or commercial conflict of interest.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results and discussion
  5. Concluding remarks
  6. Materials and methods
  7. Acknowledgements
  8. References
  9. Supporting Information
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Supporting Information

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results and discussion
  5. Concluding remarks
  6. Materials and methods
  7. Acknowledgements
  8. References
  9. Supporting Information

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