In vitro-generated MART-1-specific CD8 T cells display a broader T-cell receptor repertoire than ex vivo naïve and tumor-infiltrating lymphocytes.

The differentiation of human hematopoietic stem cells into CD8 T cells can be achieved in vitro with the OP9-DL4 system. We considered that in the absence of medullary thymic epithelial cells, which serve to restrict the breath of the T-cell receptor (TCR) repertoire by expressing tissue-restricted antigens, a distinct repertoire would be generated in vitro. To test this notion, we compared the TCR-Vα/Vß (TRAV/TRBV) gene usage of major histocompatibility complex-restricted antigen (MART-1)-specific T cells generated in vitro to that from ex vivo naïve T cells and tumor-infiltrating lymphocytes (TILs) using high-throughput DNA sequencing. In contrast to naïve T cells and TILs, which showed the expected narrow TRAV repertoire, in vitro-generated MART-1-specific T cells used almost all TRAV gene families and displayed unique CDR3 lengths. Our work demonstrates that the OP9-DL4 system supports the creation of a broad antigen-specific TCR repertoire, suggesting that T cells generated in vitro may undergo a different set of selection events that otherwise constrains the TCR repertoire of thymus-derived T cells.

Generation and molecular recognition of melanoma-associated antigen-specific human γδ T cells.

Antigen recognition by T cells bearing αß T cell receptors (TCRs) is restricted by major histocompatibility complex (MHC). However, how antigens are recognized by T cells bearing γδ TCRs remains unclear. Although γδ T cells can recognize nonclassical MHC, it is generally thought that recognition of antigens is not MHC restricted. Here, we took advantage of an in vitro system to generate antigen-specific human T cells and show that melanoma-associated antigens, MART-1 and gp100, can be recognized by γδ T cells in an MHC-restricted fashion. Cloning and transferring of MART-1-specific γδ TCRs restored the specific recognition of the initial antigen MHC/peptide reactivity and conferred antigen-specific functional responses. A crystal structure of a MART-1-specific γδ TCR, together with MHC/peptide, revealed distinctive but similar docking properties to those previously reported for αß TCRs, recognizing MART-1 on HLA-A*0201. Our work shows that antigen-specific and MHC-restricted γδ T cells can be generated in vitro and that MART-1-specific γδ T cells can also be found and cloned from the naïve repertoire. These findings reveal that classical MHC-restricted human γδ TCRs exist in the periphery and have the potential to be used in developing of new TCR-based immunotherapeutic approaches.

A monoclonal antibody against the extracellular domain of mouse and human epithelial V-like antigen 1 reveals a restricted expression pattern among CD4- CD8- thymocytes.

Expression of transcripts for the homotypic adhesion protein epithelial V-like antigen 1 (EVA1), also known as myelin protein zero like-2 (Mpzl2), is known to be present in thymic stromal cells. However, protein expression within different thymic subsets, stromal and/or lymphoid, has not been characterized due a lack of specific reagents. To address this, we generated a hybridoma (G9P3-1) secreting a monoclonal antibody (G9P3-1Mab), reactive against both human and mouse EVA1. The G9P3-1Mab was generated by immunizing Mpzl2-deficient gene-targeted mice with the extracellular domain of EVA1, followed by a conventional hybridoma fusion protocol, illustrating the feasibility of using gene-targeted mice to generate monoclonal antibodies with multiple species cross-reactivity. We confirmed expression of EVA1 on cortical and medullary epithelial cell subsets and revealed a restricted pattern of expression on CD4- CD8- double negative (DN) cell subsets, with the highest level of expression on DN3 (CD44(low)CD25(+)) thymocytes. G9P3-1MAb is a valuable reagent to study thymic T cell development and is likely useful for the analysis of pathological conditions affecting thymopoiesis, such as thymic involution caused by stress or aging.

Notch signals are required for in vitro but not in vivo maintenance of human hematopoietic stem cells and delay the appearance of multipotent progenitors.

All blood cell lineages start from hematopoietic stem cells (HSCs), which were recently shown to represent a heterogeneous group of cells. In mice, Notch signaling promotes the maintenance of "stemness" as well as the expansion of self-renewing HSCs in vitro. Additionally, human CD34(+) cells were shown to expand in vitro in response to Notch signals. However, it is unclear whether Notch directly affects all HSCs, and whether this role is relevant in vivo. Here, we developed culture conditions that support the maintenance of CD34(+)CD133(+)CD90(low)CD38(-)CD7(-)CD10(-)CD45RA(-) (CD90(low)) cells, phenotypically defined HSCs, as well as 2 early progenitor cells (CD34(+)CD38(-)CD7(-)CD10(-)CD45RA(int) [RA(int)] and CD34(+)CD38(-)CD7(-)CD10(-)CD45RA(hi) [RA(hi)]) that were functionally equivalent to multipotent progenitor-2 and lymphoid-primed multipotent progenitor, respectively, found in cord blood. Using a genetic approach, we show that Notch signals were required for HSC preservation, with cultured HSCs being equal to ex vivo HSC cells in their ability to reconstitute immunodeficient mice; however, dnMaml-transduced HSCs were not maintained in vitro. Interestingly, Notch signaling did not appear to be required for the self-renewal of human HSCs in vivo. Our findings support the notion that Notch signals maintain human HSCs in vitro that have hematopoietic-reconstituting ability in vivo and delay the appearance of 2 newly described early progenitor cells.

Human proT-cells generated in vitro facilitate hematopoietic stem cell-derived T-lymphopoiesis in vivo and restore thymic architecture.

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, CD34(+)CD7(+)CD5(-); proT2, CD34(+)CD7(+)CD5(+)) 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 κb (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 cell-based therapies to aid in the recovery of T-cells in patients undergoing HSCT.

GATA-3 regulates the self-renewal of long-term hematopoietic stem cells.

The transcription factor GATA-3 is expressed and required for differentiation and function throughout the T lymphocyte lineage. Despite evidence it may also be expressed in multipotent hematopoietic stem cells (HSCs), any role for GATA-3 in these cells has remained unclear. Here we found GATA-3 was in the cytoplasm in quiescent long-term stem cells from steady-state bone marrow but relocated to the nucleus when HSCs cycled. Relocation depended on signaling via the mitogen-activated protein kinase p38 and was associated with a diminished capacity for long-term reconstitution after transfer into irradiated mice. Deletion of Gata3 enhanced the repopulating capacity and augmented the self-renewal of long-term HSCs in cell-autonomous fashion without affecting the cell cycle. Our observations position GATA-3 as a regulator of the balance between self-renewal and differentiation in HSCs that acts downstream of the p38 signaling pathway.

Neurokinin-1 receptor signalling impacts bone marrow repopulation efficiency.

Tachykinins are a large group of neuropeptides with both central and peripheral activity. Despite the increasing number of studies reporting a growth supportive effect of tachykinin peptides in various in vitro stem cell systems, it remains unclear whether these findings are applicable in vivo. To determine how neurokinin-1 receptor (NK-1R) deficient hematopoietic stem cells would behave in a normal in vivo environment, we tested their reconstitution efficiency using competitive bone marrow repopulation assays. We show here that bone marrow taken from NK-1R deficient mice (Tacr1(-/-)) showed lineage specific B and T cell engraftment deficits compared to wild-type competitor bone marrow cells, providing evidence for an involvement of NK-1R signalling in adult hematopoiesis. Tachykinin knockout mice lacking the peptides SP and/or HK-1 (Tac1 (-/-), Tac4 (-/-) and Tac1 (-/-)/Tac4 (-/-) mice) repopulated a lethally irradiated wild-type host with similar efficiency as competing wild-type bone marrow. The difference between peptide and receptor deficient mice indicates a paracrine and/or endocrine mechanism of action rather than autocrine signalling, as tachykinin peptides are supplied by the host environment.

Targeted deletion of the tachykinin 4 gene (TAC4-/-) influences the early stages of B lymphocyte development.

Hemokinin-1 (HK-1), encoded by the TAC4 gene, is a tachykinin peptide that is predominantly expressed in non-neuronal cells, such as immune cells. We have disrupted the mouse TAC4 gene to obtain a better understanding of the actions of HK-1 during hematopoiesis. We demonstrate here that TAC4(-/-) mice exhibit an increase of CD19(+)CD117(+)HSA(+)BP.1(-) "fraction B" pro-B cells in the bone marrow, whereas pre-B, immature, and mature B cells are within the normal range. We show that in vitro cultures derived from TAC4(-/-) bone marrow, sorted "fraction B" pro-B cells or purified long-term reconstituting stem cells, contain significantly higher numbers of pro-B cells compared with controls, suggesting an inhibitory role for HK-1 on developing B cells. Supporting this idea, we show that addition of HK-1 to cultures established from long-term reconstituting stem cells and the newly described intermediate-term reconstituting stem cells leads to a significant decrease of de novo generated pro-B cells. Based on our studies, we postulate that HK-1 plays an inhibitory role in hematopoiesis, and we hypothesize that it may be part of the bone marrow microenvironment that supports and regulates the proliferation and differentiation of hematopoietic cells.

Intermediate-term hematopoietic stem cells with extended but time-limited reconstitution potential.

Sustained blood cell production depends on divisions by hematopoietic stem cells (HSCs) that yield both differentiating progeny as well as new HSCs via self-renewal. Differentiating progeny remain capable of self-renewal, but only HSCs sustain self-renewal through successive divisions securely enough to maintain clones that persist life-long. Until recently, the first identified next stage consisted of "short-term" reconstituting cells able to sustain clones of differentiating cells for only 4-6 weeks. Here we expand evidence for a numerically dominant "intermediate-term" multipotent HSC stage in mice whose clones persist for 6-8 months before becoming extinct and that are separable from both short-term as well as permanently reconstituting "long-term" HSCs. The findings suggest that the first step in stem cell differentiation consists not in loss of initial capacity for serial self-renewal divisions, but rather in loss of mechanisms that stabilize self-renewing behavior throughout successive future stem cell divisions.

Hematopoietic stem cells engraft in mice with absolute efficiency.

The engraftment of murine hematopoietic stem cells (HSCs) into irradiated mice is thought to be an inefficient process, but has yet to be measured directly. We used two independent strategies to test their engraftment efficiency: one measured competition of unpurified donor bone marrow cells with recipient cells in murine hosts and the other tracked the engraftment of one highly purified stem cell injected per recipient. The results showed that stem cells engrafted with near absolute efficiency. Thus, inefficient engraftment cannot explain the low frequency of permanent reconstitutions observed with pure HSC fractions and instead suggests most initially engrafted cells fail to sustain self-renewal.

Hematopoietic competence is a rare property of neural stem cells that may depend on genetic and epigenetic alterations.

The concept of stem-cell plasticity received strong support from a recent observation that extensively passaged, clonally derived neural stem cells could contribute to hematopoiesis. We investigated whether hematopoietic potential was a consistent or unusual feature of neural stem cells, and whether it depended on the extent of in vitro passaging before transplantation. Here we transplanted over 128 x 10(6) neurosphere cells into 128 host animals; however, we never observed contribution to hematopoiesis, irrespective of the number of passages and despite the use of an assay that could detect the contribution of a single blood stem cell to hematopoietic repopulation. Although extensively cultured neurosphere cells continued to generate neural progeny, marked changes in their growth properties occurred, including changes in growth-factor dependence, cell-cycle kinetics, cell adhesion and gene expression. Our results exclude hematopoietic competence as a consistent property of intravenously infused neural stem cells. However, the consistent changes that occurred during extended passaging are compatible with genetic or epigenetic alterations and suggest that rare transformation events may account for the neural-to-blood fate switch originally reported.