scholarly journals Artificial thymic organoids represent a reliable tool to study T-cell differentiation in patients with severe T-cell lymphopenia

2020 ◽  
Vol 4 (12) ◽  
pp. 2611-2616 ◽  
Author(s):  
Marita Bosticardo ◽  
Francesca Pala ◽  
Enrica Calzoni ◽  
Ottavia M. Delmonte ◽  
Kerry Dobbs ◽  
...  
Keyword(s):  
Cell Differentiation ◽  
T Cell ◽  
T Cell Development ◽  
Cell Development ◽  
T Cell Differentiation ◽  
Cd8 Cells ◽  
Cd34 Cells ◽  
T Cell Lymphopenia ◽  
Stromal Cell Line

Abstract The study of early T-cell development in humans is challenging because of limited availability of thymic samples and the limitations of in vitro T-cell differentiation assays. We used an artificial thymic organoid (ATO) platform generated by aggregating a DLL4-expressing stromal cell line (MS5-hDLL4) with CD34+ cells isolated from bone marrow or mobilized peripheral blood to study T-cell development from CD34+ cells of patients carrying hematopoietic intrinsic or thymic defects that cause T-cell lymphopenia. We found that AK2 deficiency is associated with decreased cell viability and an early block in T-cell development. We observed a similar defect in a patient carrying a null IL2RG mutation. In contrast, CD34+ cells from a patient carrying a missense IL2RG mutation reached full T-cell maturation, although cell numbers were significantly lower than in controls. CD34+ cells from patients carrying RAG mutations were able to differentiate to CD4+CD8+ cells, but not to CD3+TCRαβ+ cells. Finally, normal T-cell differentiation was observed in a patient with complete DiGeorge syndrome, consistent with the extra-hematopoietic nature of the defect. The ATO system may help determine whether T-cell deficiency reflects hematopoietic or thymic intrinsic abnormalities and define the exact stage at which T-cell differentiation is blocked.

Constitutive Expression of MEF2C, LYL1, or LMO2 in CD34+ HSCs Blocks the in Vitro Differentiation before the T-Cell Commitment Point and Could be Oncogenic in Early T-Cell Progenitor ALL

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 2423-2423
Author(s):  
Kirsten Canté-Barrett ◽  
Rui D Mendes ◽  
Wilco K Smits ◽  
Rob Pieters ◽  
Jules PP Meijerink
Keyword(s):  
Gene Expression ◽  
T Cells ◽  
Cell Differentiation ◽  
T Cell ◽  
T Cell Development ◽  
Cell Development ◽  
T Cell Differentiation ◽  
Cell Commitment

Abstract Background: T-cell development in the thymus is a complex process that depends on sequential transcriptional and epigenetic events that induce T-cell lineage commitment and simultaneously suppress alternative cell fates. In T-cell acute lymphoblastic leukemia (T-ALL), aberrantly expressed oncogenes result in the arrest of developing thymocytes, which can lead to the acquisition of secondary mutations, uncontrolled proliferation and disease progression. MEF2C is often expressed as a result of chromosomal rearrangements in immature, early T-cell progenitor ALL (ETP-ALL), but is also expressed in normal thymocyte progenitors before T-cell commitment (in the ETP stage). As the only hematopoietic lineage, thymocytes that have passed the T-cell commitment checkpoint (as well as mature T-cells) do no longer express MEF2C. Aims: We aimed to investigate the effect of constitutive MEF2C expression on early T-cell development. OP9-DL1 co-cultures have been most useful for mimicking in vitro T-cell development starting with hematopoietic stem cells (HSCs) derived from human cord blood or bone marrow. We also aimed to investigate the impact of MEF2C in comparison to LYL1 and LMO2; two T-ALL oncogenes also highly expressed at the ETP stage. Methods: We have utilized the OP9-DL1 in vitro co-culture system to gradually differentiate CD34+ HSCs from umbilical cord blood into the T-cell lineage. HSCs in this co-culture will recapitulate in vivo T-cell development as measured by incremental acquisition of surface markers CD7, CD5, CD1a, and reach the CD4, CD8 double-positive (DP) stage. We generated gene expression profiles of 11 subsequent in vitro stages of differentiation to help us match them to in vivo development stages. We investigated in vitro T-cell differentiation of HSCs after lentiviral transduction with MEF2C or control vectors, as well as with other transcriptional regulators LYL1 and LMO2 that are expressed at the ETP stage. Results: The major change in gene expression of subsequent early T-cell differentiation stages defines two distinct T-cell differentiation clusters that correlate with in vivo pre- and post-T-cell commitment profiles. We found that T-cell commitment occurs in CD7+ CD5+ cells before the acquisition of CD1a surface expression. Expression of control vectors in HSCs does not affect the in vitro T-cell differentiation, but MEF2C expression blocks differentiation into the direction of T-cells as measured by the failure of most cells to acquire CD7 as the first marker. Instead, with increased passage number cells gradually lose CD34 expression and eventually disappear from the co-culture. Similar effects were observed for the expression of LYL1 and LMO2; LYL1 expression arrests the cells at the most immature CD7+ ETP stage and prevents the transition towards CD7+ CD5+ cells, whereas LMO2 expressing cells reach the CD7+ CD5+ stage but fail to acquire CD1a as a marker of T-cell commitment. Summary/Conclusion: The gene expression profiles of 11 human in vitro T-cell differentiation subsets has enabled us to pinpoint T-cell commitment to a stage in which cells have acquired CD7 and CD5, just prior to the acquisition of CD1a. MEF2C, LYL1, and LMO2, expressed in ETP-ALL as well as in normal thymocyte progenitors, do not allow the transition to T-cell commitment when constitutively expressed. These proteins each result in the arrest of in vitro differentiating T-cells at different ETP stages, all before the T-cell commitment as marked by CD1a expression. Constitutive expression of MEF2C, LYL1, or LMO2 in very early thymocyte progenitors is incompatible with development into and beyond the T-cell commitment checkpoint and these proteins could therefore play important roles in the pathogenesis of ETP-ALL. Disclosures No relevant conflicts of interest to declare.

Opposing Effects of Toll-Like Receptor Ligands and Ascorbic Acid On T and Natural Killer Cell Development From Lymphoid Progenitor Cells.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 1491-1491 ◽  
Author(s):  
Birgitta Mitchell ◽  
Maritza Gonzalez ◽  
Jared Manning ◽  
Gerald J Spangrude
Keyword(s):  
Ascorbic Acid ◽  
Cell Differentiation ◽  
T Cell ◽  
Progenitor Cells ◽  
Nk Cell ◽  
Cell Receptor ◽  
Cell Development ◽  
T Cell Differentiation ◽  
Nk Cell Differentiation

Abstract Abstract 1491 Poster Board I-514 Introduction: A complete understanding of lymphocyte development, particularly factors driving T and natural killer (NK) cell differentiation from progenitor cells, remains an elusive goal in medicine. T and NK cells are key regulators in the defense against infections and malignancies and play a direct causative role in autoimmune diseases and graft-versus-host disease. The OP9-DL1 stromal line is an important tool in the in vitro study of lymphocyte development. Lymphocyte progenitors (KLS,Thy1.1-) harvested from adult murine bone marrow and seeded on this stromal line can be followed through stages of maturation by immunophenotyping. We observed that addition of stem cell factor (SCF), contaminated with lipopolysaccharide (LPS) through its production in E. coli, was particularly effective at promoting NK cell development in the OP9-DL1 culture system. Toll-like receptors, an important component of anti-microbial defense by the innate immune response, recognize LPS and other microbial products. Toll-like receptor ligands (TLR-L) have been shown to enhance NK cell proliferation, however an effect on NK cell differentiation from progenitor cells has not been established. A separate set of experiments led us to hypothesize that ascorbic acid (vitamin C) promotes T cell differentiation. We therefore designed experiments to evaluate the differential effects of TLR-L and ascorbic acid on NK and T cell development from lymphoid progenitors co-cultured with OP9-DL1 stromal cells. Methods: Lymphocyte progenitor cells (KLS,Thy1.1-) were sorted from adult mouse bone marrow and 1000-2000 progenitor cells were seeded per well in a 24 well plate coated with OP9-DL1 stroma. Cultures were supplemented with IL-7 (5 ng/ml), Flt3 ligand (5 ng/ml), and SCF (100 ng/ml) plus one of 5 different TLR-L (TLR1/2, TLR3, TLR4, TLR5, and a crude LPS preparation that likely contains a number of TLR-L), with or without addition of a stabilized form of ascorbic acid. Cells were passaged, counted and re-seeded with fresh media and supplements twice a week over a 30-day period. Immunophenotype and viability were evaluated by flow cytometry. Markers for T cell development included CD44, CD25, CD3, CD4, CD8, T cell receptor beta chain and T cell receptor gamma-delta chains. NK cells were evaluated for the presence of NKp46, NK1.1, and DX5. Results: We observed robust cell expansion, inhibited somewhat by addition of ascorbic acid. The inhibitory effect of ascorbate on expansion was most pronounced in the culture condition lacking TLR-L. T cell differentiation was markedly advanced by the addition of ascorbic acid in the absence of TLR-L, with the majority of cells co-expressing CD4/CD8 and TCRB/CD3. The addition of different TLR-Ls inhibited T cell differentiation, and this inhibition was partially rescued by addition of ascorbic acid. NK cell differentiation, defined as co-expression of NKp46 and NK1.1, was two to three-fold greater with the addition of TLR1/2, TLR4, TLR5, and crude LPS compared to cultures lacking TLR-L addition. In each of these conditions, NK cell differentiation was markedly inhibited by addition of ascorbic acid. Conclusions: Our data supports the hypothesis that both T and NK cell progenitors require Notch signaling for differentiation. In our in vitro model, differentiation of one lineage at the expense of the other can be manipulated with addition of TLR-L or ascorbic acid. Addition of bacterial TLR-L promotes NK cell differentiation at the expense of T cell differentiation; an effect that is partially overcome with the addition of ascorbic acid. The addition of ascorbic acid promotes robust T cell differentiation, and inhibits significant NK cell differentiation in all conditions. The ability of ascorbic acid to promote T cell differentiation appears to dominate over TLR-L promotion of NK lineage differentiation. Further work will include microarray to evaluate these effects at a genetic level. These findings will contribute to our understanding of the immune response under normal and pathologic conditions, and further a model both for study and ex vivo expansion of immune cells for therapeutic use. Disclosures: No relevant conflicts of interest to declare.

scholarly journals In vitro T lymphopoiesis of human and rhesus CD34+ progenitor cells

Blood ◽  
1996 ◽  
Vol 87 (10) ◽  
pp. 4040-4048 ◽  
Author(s):  
M Rosenzweig ◽  
DF Marks ◽  
H Zhu ◽  
D Hempel ◽  
KG Mansfield ◽  
...  
Keyword(s):  
T Cells ◽  
Cell Differentiation ◽  
T Cell ◽  
T Lymphocytes ◽  
Progenitor Cells ◽  
T Cell Differentiation ◽  
Hematopoietic Stem ◽  
Human Cd34 ◽  
Cd34 Cells

Differentiation of hematopoietic progenitor cells into T lymphocytes generally occurs in the unique environment of the thymus, a feature that has hindered efforts to model this process in the laboratory. We now report that thymic stromal cultures from rhesus macaques can support T-cell differentiation of human or rhesus CD34+ progenitor cells. Culture of rhesus or human CD34+ bone marrow-derived cells depleted of CD34+ lymphocytes on rhesus thymic stromal monolayers yielded CD3+CD4+CD8+, CD3+CD4+CD8-, and CD3+CD4-CD8+ cells after 10 to 14 days. In addition to classical T lymphocytes, a discrete population of CD3+CD8loCD16+CD56+ cells was detected after 14 days in cultures inoculated with rhesus CD34+ cells. CD3+ T cells arising from these cultures were not derived from contaminating T cells present in the CD34+ cells used to inoculate thymic stromal monolayers or from the thymic monolayers, as shown by labeling of cells with the lipophilic membrane dye PKH26. Expression of the recombinase activation gene RAG- 2, which is selectively expressed in developing lymphocytes, was detectable in thymic cultures inoculated with CD34+ cells but not in CD34+ cells before thymic culture or in thymic stromal monolayers alone. Reverse transcriptase-polymerase chain reaction analysis of T cells derived from thymic stromal cultures of rhesus and human CD34+ cells showed a polyclonal T-cell receptor repertoire. T-cell progeny derived from rhesus CD34+ cells cultured on thymic stroma supported vigorous simian immunodeficiency virus replication in the absence of exogenous mitogenic stimuli. Rhesus thymic stromal cultures provide a convenient means to analyze T-cell differentiation in vitro and may be useful as a model of hematopoietic stem cell therapy for diseases of T cells, including acquired immunodeficiency syndrome.

In Vitro Exposure to DL-4 Increases the in Vitro and In Vivo T-Lymphopoietic Potential of CB Derived CD34+ Progenitor Cells

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2980-2980
Author(s):  
Christian Reimann ◽  
Liliane Liliane Dal-Cortivo ◽  
Emmanuelle M. Six ◽  
Andrea Schiavo ◽  
Marina Cavazzana-Calvo ◽  
...  
Keyword(s):  
T Cell ◽  
Developmental Stages ◽  
T Cell Development ◽  
Cell Development ◽  
T Cell Reconstitution ◽  
Cd34 Cells ◽  
Peripheral T Cells ◽  
Lymphoid Progenitors

Abstract Abstract 2980 Notchligand-based culture systems such as OP9-DL1 cells induce HSC to engage towards the T-cell developmental program and allow generation of T-lymphoid progenitors in vitro. In vitro generated murine T-lymphoid progenitors accelerated T-cell reconstitution in vivo. In consistency, human T-lymphoid progenitors generated in co-culture with OP9-DL1 cells enhanced thymic repopulation when injected into NOD/SCID/gc−/− mice (NSG). However, positive effects of human T-lymphoid progenitors on peripheral T-cell reconstitution have not been reported yet. Besides, Notchligand-based culture systems, consisting of genetically modified murine cells might raise safety concern for clinical use. It has been described that exposure of CD34+ cells to immobilized DL4 induces the T-cell developmental program even in absence of stromal cell support. Recently, we have made use of this system to generate T-lymphoid progenitors in vitro. In the present study we have further characterized their T-lymphoid potential in vitro and in vivo. Exposure of human CB-derived CD34+ cells to immobilized DL4 allowed generation of CD34+CD7+ and CD34−CD7++CD5+ progenitors displaying a similar phenotype as early thymic progenitors (ETP) and the prethymocytes (pre-T). Within the DL-4 derived ETP- and preT-like progenitors we observed subsequent up regulation of genes involved in T-cell development and silencing of genes implied in B-cell and myeloid differentiation. T-cell commitment of DL-4 progenitors could be further confirmed by early and intermediate rearrangement events within the TCR d/g/b genes. The pattern of gene expression profile and TCR-rearrangement events displayed a pattern similar to what we observed in corresponding intrathymic developmental stages. DL4-progenitors obtained after 7 days of culture displayed a 30-fold increased in vitro T-lymphoid potential as compared to untreated CD34+ CB progenitors. DL4 ETP-like and preT-like progenitors further completed T-cell differentiation in vitro (in OP9DL1 co-culture) faster than native CD34+ CB progenitors. When transferred into NSG, DL4 progenitors obtained after 7 days of culture were able to repopulate the recipients' thymus and to give rise to mature, polyclonal intrathymic and peripheral T-cells. Two months after transfer recipients of DL4 progenitors displayed advanced intrathymic T-cell development as compared to recipients of CD34+ CB cells. Furthermore, peripheral T-cells could be observed in a number of DL-4 progenitor recipients but not in control mice. Our experiments provide further evidence that DL4 allows in vitro induction of T-cell development and generation of early T-lymphoid progenitors in a system devoid of stromal cell support. These progenitors feature phenotypical and molecular characteristics of immature thymic developmental stages. Moreover, they are able to accelerate T-cell development in vitro and when transferred into NSG. This work provides further evidence of the ability of in vitro -generated human T-cell progenitors to accelerate T-cell reconstitution and simultaneously introduces a culture technique that could be rapidly transferred into a clinical setting. Disclosures: No relevant conflicts of interest to declare.

Induction of T-cell development from human cord blood hematopoietic stem cells by Delta-like 1 in vitro

Blood ◽  
2005 ◽  
Vol 105 (4) ◽  
pp. 1431-1439 ◽  
Author(s):  
Ross N. La Motte-Mohs ◽  
Elaine Herer ◽  
Juan Carlos Zúñiga-Pflücker
Keyword(s):  
Stem Cells ◽  
T Cells ◽  
Cell Differentiation ◽  
T Cell ◽  
Cord Blood ◽  
T Cell Development ◽  
T Cell Differentiation ◽  
Human Cord Blood ◽  
Hematopoietic Stem

AbstractThe Notch signaling pathway plays a key role at several stages of T-lymphocyte differentiation. However, it remained unclear whether signals induced by the Notch ligand Delta-like 1 could support full T-cell differentiation from a defined source of human hematopoietic stem cells (HSCs) in vitro. Here, we show that human cord blood–derived HSCs cultured on Delta-like 1–expressing OP9 stromal cells undergo efficient T-cell lineage commitment and sustained T-cell differentiation. A normal stage-specific program of T-cell development was observed, including the generation of CD4 and CD8 αβ–T-cell receptor (TCR)–bearing cells. Induction of T-cell differentiation was dependent on the expression of Delta-like 1 by the OP9 cells. Stimulation of the in vitro–differentiated T cells by TCR engagement induced the expression of T-cell activation markers and costimulatory receptors. These results establish an efficient in vitro coculture system for the generation of T cells from human HSCs, providing a new avenue for the study of early T-cell differentiation and function.

scholarly journals B cell adaptor for PI3-kinase (BCAP) modulates CD8+ effector and memory T cell differentiation

2018 ◽  
Vol 215 (9) ◽  
pp. 2429-2443 ◽  
Author(s):  
Mark D. Singh ◽  
Minjian Ni ◽  
Jenna M. Sullivan ◽  
Jessica A. Hamerman ◽  
Daniel J. Campbell
Keyword(s):  
T Cells ◽  
Cell Differentiation ◽  
T Cell ◽  
B Cell ◽  
Cd8 T Cells ◽  
T Cell Development ◽  
Cell Development ◽  
T Cell Differentiation ◽  
Pi3k Signaling ◽  
Memory T Cell

CD8+ T cells respond to signals via the T cell receptor (TCR), costimulatory molecules, and immunoregulatory cytokines by developing into diverse populations of effector and memory cells. The relative strength of phosphoinositide 3-kinase (PI3K) signaling early in the T cell response can dramatically influence downstream effector and memory T cell differentiation. We show that initial PI3K signaling during T cell activation results in up-regulation of the signaling scaffold B cell adaptor for PI3K (BCAP), which further potentiates PI3K signaling and promotes the accumulation of CD8+ T cells with a terminally differentiated effector phenotype. Accordingly, BCAP-deficient CD8+ T cells have attenuated clonal expansion and altered effector and memory T cell development following infection with Listeria monocytogenes. Thus, induction of BCAP serves as a positive feedback circuit to enhance PI3K signaling in activated CD8+ T cells, thereby acting as a molecular checkpoint regulating effector and memory T cell development.

Distinctive Roles of Stem Cell Factor and IL-7 in T Cell Development.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 3156-3156
Author(s):  
Hongfang Wang ◽  
L. Jeanne Pierce ◽  
Gerald J. Spangrude
Keyword(s):  
Stem Cell ◽  
Cell Differentiation ◽  
T Cell ◽  
Notch Signaling ◽  
Stem Cell Factor ◽  
Critical Role ◽  
T Cell Development ◽  
Cell Development ◽  
T Cell Differentiation ◽  
Lineage Differentiation

Abstract Notch signaling plays a critical role in T lineage commitment during lymphoid differentiation. However, Notch signaling alone is not sufficient to support T cell development through the CD4/CD8 double positive (DP) stage in vitro. We here report distinct effects of several cytokines on T cell differentiation in the OP9-DL1 cell culture model. Our studies show that Flt3 ligand enhances the proliferation of progenitors but has no obvious effect on differentiation. In contrast, stem cell factor (SCF) favors the proliferation of CD4/CD8 double negative (DN) lymphoid progenitors and inhibits differentiation to the DP stage in a dose-dependent manner. Differentiation of the NK lineage is promoted under these conditions. Conversely, blocking the function of SCF that is expressed endogenously by OP9-DL1 cells inhibits proliferation of lymphoid progenitors and accelerates T lineage differentiation. IL-7 is necessary for differentiation from the DP to the CD8 single positive (SP) stage, and is also required for γδ T lineage development. We also find a dosage effect of IL-7 during T cell development. OP9 and OP9-DL1 stromal cells produce endogenous levels of IL-7 that are sufficient to support B and DP T cell differentiation. However, the amount of endogenous IL-7 is not sufficient to support T cell differentiation from the DP to the SP stage. Addition of exogenous IL-7 (1–10 ng/ml) to the cultures promotes SP differentiation, while blocking endogenous IL-7 with anti-IL-7 antibody inhibits both B and T cell development. We conclude that activation through the Notch pathway is sufficient to suppress B lineage differentiation and thereby promote T lineage commitment, but is not sufficient to promote the subsequent stages of T cell development. SCF promotes expansion and directs NK lineage differentiation at the expense of T cell development, while IL-7 provides both proliferation as well as T lineage differentiation signals. T cell development from the DN to the DP stage requires a low amount of IL-7, while differentiation from the DP to the SP stage requires a higher level of IL-7. The balance between the effects mediated by these cytokines, along with Notch signaling, plays a critical role in regulating development of the T and NK lineages.

Dysregulation of T-Cell Development in Adrenal Glucocorticoid-Deprived Rats

2009 ◽  
Vol 234 (9) ◽  
pp. 1067-1074 ◽  
Author(s):  
Zorica Stojić-Vukanić ◽  
Aleksandra Rauški ◽  
Duško Kosec ◽  
Katarina Radojević ◽  
Ivan Pilipović ◽  
...  
Keyword(s):  
Cell Differentiation ◽  
T Cell ◽  
T Cell Development ◽  
Cell Development ◽  
T Cell Differentiation ◽  
Male Rats ◽  
Double Positive ◽  
Cell Repertoire ◽  
Single Positive ◽  
The Impact

A number of different experimental approaches have been used to elucidate the impact of basal levels of adrenal gland-derived glucocorticoids (GCs) on T cell development, and thereby T cell-mediated immune responses. However, the relevance of the adrenal GCs to T cell development is still far from clear. This study was undertaken to explore the relevance of basal levels of GCs to T cell differentiation/maturation. Eight days post-adrenalectomy in adult male rats the thymocyte yield, apoptotic and proliferative rate and the relationship amongst major thymocyte subsets, as defined by TCRαβ/CD4/CD8 expression, were examined using flow cytometry. Adrenal GC deprivation decreased thymocyte apoptosis and altered the kinetics of T cell differentiation/maturation. In the adrenalectomized rats there was increased thymic hypercellularity and an over-representation of the CD4+CD8+ double positive (DP) TCRαβlow cells entering selection, as well as increased numbers of their DP TCRαβ− immediate precursors. These changes were accompanied with under-representation of the postselected DP TCRαβhigh and the most mature CD4−CD8+ and, particularly, CD4+CD8− single positive (SP) TCRαβhigh cells. This data suggests that withdrawal of adrenal GCs produces alterations in the thymocyte selection processes, possibly affecting the diversity of functional T cell repertoire and generation of potentially self-reactive cells as indicated by the reduced proportion and number of CD4−CD8− double negative TCRαβhigh cells. In addition, it indicates that GCs influence the post-selection maturation of thymocytes and plays a regulatory role in controlling the ratio of mature CD4+CD8−/CD4−CD8+ SP TCRαβhigh cells.

In Vitro Generation of Human T-Cell Precursors From Bone Marrow CD34+ Cells by Short Exposure to Immobilized Notch-Ligand Delta-Like 4.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3532-3532
Author(s):  
Christian Reimann ◽  
Liliane Dal-Cortivo ◽  
Brigitte Ternaux ◽  
Emmanuelle Six ◽  
Julien Rouiller ◽  
...  
Keyword(s):  
T Cell ◽  
Nk Cell ◽  
T Cell Development ◽  
Cell Development ◽  
Short Exposure ◽  
Cell Phenotype ◽  
Notch Ligand ◽  
Human T Cell ◽  
Cd34 Cells

Abstract Abstract 3532 Poster Board III-469 Prolonged posttransplant immune deficiency is a major complication following hematopoietic stem cell transplantation, particularly in the T-lymphoid compartment. Accelerating T-cell development by injecting donor derived T-cell precursors has been proposed as a novel strategy to shorten the immune deficient phase. Several research groups have successfully generated T-cell precursors from murine and human HSC in vitro by transitory exposure to the Notch-ligand presenting murine OP9DL1-cell line. Transfer of the in vitro generated murine T-cell precursors into irradiated NOD/SCID/γcnull-mice accelerated T-cellular reconstitution. However, the clinical application of the OP9DL1-system is limited. Recent studies have demonstrated that short exposure of cord blood CD34+ cells to Notch-ligand Delta-like 4 is sufficient to promote human T-cell differentiation in vitro. Here, we modified this technique to better characterize and ameliorate T-cell development in vitro, with the objective of eventually transferring this method to a clinical phase. Towards this aim, we exposed human CD34+ HSC derived from any available source to immobilized Notch-ligand Delta-like 4 in the presence of different cytokine combinations implicated in human haematopoiesis (IL-7, SCF, Flt3-ligand and TPO). Within 7 days a population of CD34+CD7+ and CD34-CD7++ T-cell precursors emerged in the presence of Delta-like 4, but not under control conditions. After 7 days the CD34+CD7+ population subsequently declined while further amplification of the CD34-CD7++ population was observed. Two distinct progenitor subsets emerged within the CD34-CD7++ population, namely CD34-CD7++CD5+ and CD34-CD7++CD5-. The CD34-CD7++CD5+ subset further acquired CD1a and, thus, adopted a pre-T-cell phenotype. Between days 7 and 14 the CD34-CD7++CD5- acquired a NK-cell phenotype, as indicated by CD16 and CD56 expression. Beyond 14 days no further expansion of the pre-T-cell fraction was observed, while the NK-cell fraction continued proliferating. More advanced stages T-cell development, such as immature single positive CD4+ cells as observable in OP9DL1 co-cultures, did not arise after exposing cells only to immobilized Delta-like4. Intermittent emergence of a CD13+CD14+CD7- myeloid population was observed within the first 14 days of culture on Delta-like 4; however, this population disappeared spontaneously and did not preserve its common myeloid progenitor. Selecting a more immature CD34+CD38- population resulted in a two-fold increase of the frequency of CD34+CD7+ and CD34-CD7++ cells as compared to the whole CD34+ population, while myeloid differentiation was inhibited. A further increase was obtained by replanting cultured cells to freshly coated plates with Delta-like 4 every 3 days of culture. T-cell precursors cells derived after 7 days of culture were injected into NOD/SCID/γcnull mice. The in vivo-experiments are ongoing and results are pending. Our results provide further evidence that human T-cell precursors can be generated in vitro, not only in co-culture with murine OP9DL1-cells but also by short exposure to immobilized Notch-ligand Delta-like 4. These ongoing experiments are an important prerequisite for the potential clinical application of this method. Disclosures: No relevant conflicts of interest to declare.

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