IntroductionHematopoietic stem cells (HSCs), which give rise to erythroid, myeloid, and lymphoid lineages, can be identified on the basis of the expression of CD34 and the absence of lineage-specific markers (termed LinϪ). 1 Human umbilical cord blood (CB)-derived cells provide a rich source of HSCs, which are comparable to bone marrow-derived HSCs. [2][3][4][5][6][7][8][9][10][11] T cells of the ␣␤ lineage differentiate in the thymus via discrete developmentally regulated steps that involve a series of commitment events and developmental checkpoints including T-cell receptor (TCR) V(D)J rearrangement, TCR-␤-selection, and positive/negative selection of developing thymocytes. 12 The earliest intrathymic progenitors express high levels of CD34 and CD7, do not express CD1a, and are triplenegative (TN) for mature T-cell markers CD4, CD8, and CD3. 8 Commitment to the T-cell lineage is strongly associated with the expression of CD1a by CD7-expressing thymocytes. 12,13 Several studies have implicated the Notch pathway in promoting HSC expansion, self-renewal, 14 survival, 15,16 and the induction of T-cell-lineage commitment. 15,[17][18][19][20][21] In humans there are 4 Notch receptors, 22-26 which can pair with 2 serrate-like ligands (Jagged 1 and 2) 27,28 or 3 Delta-like ligands (Dll1, 3, and 4). 29,30 Notch signaling acts at multiple stages during T-cell differentiation, influencing the choice to become an ␣␤-versus ␥␦-T cell, 31-34 as well as the decision to become a CD4 versus CD8 T cell. [35][36][37] The strongest evidence for the role of Notch signaling in T-cell development comes from gain-of-function and loss-of-function studies, 17,20,[38][39][40][41][42] in which signaling through Notch-1 was shown to play a crucial role in determining B-versus T-cell-lineage choice. 20,21 HSCs express multiple Notch receptors, 22,43 but the expression patterns of the various Notch ligands have been reported to be distinct between bone marrow stromal cells 29,[44][45][46][47] and thymic epithelial cells. 48 Taken together, these results suggest that different Notch receptors and ligands may control different aspects of hematopoiesis, depending on the microenvironment, allowing for self-renewal in the bone marrow and influencing cell fate decisions in the thymus. 46 This led us to hypothesize that bone marrow stromal lines, such as OP9 cells, 49 that support B-cell differentiation may do so because the appropriate Notch ligand to induce T-cell commitment and differentiation is absent. We recently demonstrated that the OP9 bone marrow stromal cell line, which does not express the Delta-like 1 (Dll1) Notch ligand, when retrovirally transduced to express Dll1 (OP9-DL1), inhibited the development of B cells and rather favored the development of T cells from fetal liver-derived HSCs 50 or mouse embryonic stem (ES) cells. 51 Given the high level of homology between mouse and human Dll1 molecules and the observation that mouse stromal lines can support the differentiation of human HSCs, 29,52-54 we sought to determine whether human...

T-cell development follows a defined set of stage-specific differentiation steps. However, molecular and cellular events occurring at early stages of human T-cell development remain to be fully elucidated. To address this, human umbilical cord blood (UCB) hematopoietic stem cells (HSCs) were induced to differentiate to the T lineage in OP9-DL1 cocultures. A developmental program involving a sequential and temporally discrete expression of key differentiation markers was revealed. Quantitative clonal analyses demonstrated that CD34 ؉ CD38 ؊ and CD34 ؉ CD38 lo subsets of UCB contain a similarly high Tlineage progenitor frequency, whereas the frequency in CD34 ؉ CD38 ؉/hi cells was 5-fold lower. Delta-like/Notch-induced signals increased the T-cell progenitor frequency of CD34 ؉ CD38 ؊/lo cells differentiated on OP9-DL1, and 2 distinct progenitor subsets, CD34 ؉ CD45RA ؉ CD7 ؉؉ CD5 ؊ CD1a ؊ (proT1) and CD34 ؉ CD45RA ؉ CD7 ؉؉ CD5 ؉ CD1a ؊ (proT2), were identified and their thymus engrafting capacity was examined, with proT2 cells showing a 3-fold enhanced reconstituting capacity compared with the proT1 subset. Furthermore, in vitrogenerated CD34 ؉ CD7 ؉؉ progenitors effectively engrafted the thymus of immunodeficient mice, which was enhanced by the addition of an IL-7/IL-7 antibody complex. Taken IntroductionT cells develop within the thymus from bone marrow-derived hematopoietic progenitors, and follow a series of stage-specific differentiation events, which are broadly characterized by the developmentally coordinated expression of CD4 and CD8. 1 The initial stages of human T-cell development include precursors that express the stem cell marker CD34, 2,3 which is also present on hematopoietic stem cells (HSCs) and on multipotent or lineage-specified progenitor cells. Within the known hierarchy of T-cell development, the earliest precursor subset is further defined by the lack of CD3, CD4, CD8, and CD1a expression. 4 Although immature stages of T-cell development are typically delineated as CD34 ϩ CD1a Ϫ and CD34 ϩ CD1a ϩ cells, these populations remain heterogeneous. Of note, CD7 expression is considered to be one of the earliest cell surface markers known to appear during T lymphopoiesis. 2,5 Importantly, the transition from CD34 ϩ CD7 ϩ CD1a Ϫ to CD34 ϩ CD7 ϩ CD1a ϩ by early thymocytes is associated with T-cell commitment, as a small percentage (ϳ 10%) of these cells show rearrangement at the T-cell receptor ␤-chain (TCR␤) locus. 6 In addition, CD34 ϩ CD7 ϩ CD1a ϩ cells appear to be T-lineage restricted, as these cells show low precursor activity toward non-T-cell lineages. 7 Current understanding of the aforementioned early stages has been obtained from analyses of human fetal or adult thymocyte subsets, and by analyzing T-cell development in vitro using xenogeneic engraftment of mouse fetal thymus organ cultures (FTOCs). 8,9 Although these systems have provided important insight into T-cell development, the capacity to evaluate specific progenitor populations has remained difficult to assess given the requirem...

• Intrathymic T-cell regeneration is facilitated by human proTcells generated in vitro.• In vitro-generated human proT-cells home to the thymus, wherein they restore thymic structure.Hematopoietic stem cell transplantation (HSCT) is followed by a period of immune deficiency due to a paucity in T-cell reconstitution. Underlying causes are a severely dysfunctional thymus and an impaired production of thymus-seeding progenitors in the host. Here, we addressed whether in vitro-derived human progenitor T (proT)-cells could not only represent a source of thymus-seeding progenitors, but also able to influence the recovery of the thymic microenvironment. We examined whether co-transplantation of in vitro-derived human proT-cells with hematopoietic stem cells (HSCs) was able to facilitate HSC-derived T-lymphopoiesis posttransplant. A competitive transfer approach was used to define the optimal proT subset capable of reconstituting immunodeficient mice. Although the 2 subsets tested (proT1,1 ) showed thymus engrafting function, proT2-cells exhibited superior engrafting capacity. Based on this, when proT2-cells were coinjected with HSCs, a significantly improved and accelerated HSC-derived T-lymphopoiesis was observed. Furthermore, we uncovered a potential mechanism by which receptor activator of nuclear factor kb (RANK) ligand-expressing proT2-cells induce changes in both the function and architecture of the thymus microenvironment, which favors the recruitment of bone marrow-derived lymphoid progenitors. Our findings provide further support for the use of Notch-expanded progenitors in cellbased therapies to aid in the recovery of T-cells in patients undergoing HSCT. (Blood. 2013;122(26):4210-4219)

BackgroundT cell development occurs within the highly specialized thymus. Cytotoxic CD8 T cells are critical in adaptive immunity by targeting virally infected or tumor cells. In this study, we addressed whether functional CD8 T cells can be generated fully in vitro using human umbilical cord blood (UCB) hematopoietic stem cells (HSCs) in coculture with OP9-DL1 cells.ResultsHSC/OP9-DL1 cocultures supported the differentiation of CD8 T cells, which were TCR/CD3hi CD27hi CD1aneg and thus phenotypically resembled mature functional CD8 single positive thymocytes. These in vitro-generated T cells also appeared to be conventional CD8 cells, as they expressed high levels of Eomes and low levels of Plzf, albeit not identical to ex vivo UCB CD8 T cells. Consistent with the phenotypic and molecular characterization, upon TCR-stimulation, in vitro-generated CD8 T cells proliferated, expressed activation markers (MHC-II, CD25, CD38), secreted IFN-γ and expressed Granzyme B, a cytotoxic T-cell effector molecule.ConclusionTaken together, the ability to direct human hematopoietic stem cell or T-progenitor cells towards a mature functional phenotype raises the possibility of establishing cell-based treatments for T-immunodeficiencies by rapidly restoring CD8 effector function, thereby mitigating the risks associated with opportunistic infections.

Traditionally, the study of human T cell development has relied on the availability of human and mouse thymic tissue. In this chapter, we outline a simple in vitro protocol for generating large numbers of human T-lineage cells from umbilical cord blood (CB)- derived hematopoietic stem cells (HSCs) using a bone marrow stromal cell line. This protocol is broken into three major steps: (1) the maintenance of a working stock of OP9 bone marrow stromal cells expressing the Notch receptor ligand Delta-like 1 (OP9- DL1), (2) the purification of human HSCs from umbilical CB, and (3) the initiation and maintenance/expansion of OP9-DL1 cocultures over time (see Fig. 1). The use of this system opens avenues for basic research as it equips us with a simple in vitro method for studying human T cell development.

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T cells develop within the unique microenvironment provided by the thymus. T cell differentiation involves a series of commitment events and developmental checkpoints including T cell receptor (TCR) gene recombination and positive/negative selection of developing thymocytes to yield functionally mature T cells. These events occur in a sequential, temporal and spatial fashion, as developing thymocytes migrate through the thymus. In vitro studies to yield insights into human T cell development have classically employed the fetal thymic organ culture (FTOC) model system. This approach relies on the seeding of human hematopoietic stem cells (HSCs) and/or their progeny into host thymic lobes or thymic fragments, typically of mouse origin. Recently, a novel in vitro approach that makes use of the OP9 bone marrow stromal cell line expressing the Notch receptor ligand Delta-like-1 (OP9-DL1) effectively supported the generation of large numbers of human progenitor T cells from HSCs. In this review, we outline several in vitro systems employed for the generation and study of human T cells. Particular emphasis is dedicated to the OP9-DL1 coculture system. Finally, given the number of progenitor T cells that can be generated in vitro, we discuss the potential implications for the treatment of immune-related diseases such as cancer, immunedeficiency, and autoimmunity.