Ross N. La Motte-Mohs

The role of nuclear factor-κB essential modulator (NEMO) in B cell development and survival

The transcription factor nuclear factor-κB (NF-κB) is essential for immune and inflammatory responses. NF-κB essential modulator (NEMO) is a scaffolding component of the IκB kinase complex required for NF-κB activation in vitro. Because NF-κB activation is involved in B cell development and function, we set out to determine whether NEMO is required for these processes. NEMO(−/−) mice die very early during embryogenesis, and fetal livers from NEMO(−/−) embryos can not reconstitute either B or T lymphopoiesis in irradiated host mice. We therefore used NEMO(−/−) embryonic stem cells and the OP9 in vitro differentiation system to demonstrate that NEMO is not required for B cell development but plays an important role in B cell survival.

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.

Human CD8 T cells generated in vitro from hematopoietic stem cells are functionally mature

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.

In Vitro Human T Cell Development Directed by Notch–Ligand Interactions

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.

TLR Ligand-Induced Type I IFNs Affect Thymopoiesis

The interactions between TLRs and their ligands have profound immune modulation properties. Attention has focused mostly on the impact of TLR ligands on peripheral innate and adaptive immunity during viral infections, whereas little impact of TLR activation has been shown on thymic development. Here we show that treatment of murine fetal thymic organ cultures (FTOCs) with TLR3 or TLR7 ligands induced rapid expression of IFN-α and -β mRNA, hallmarks of acute and chronic viral infections. This resulted in an early developmental blockade, increased frequencies of apoptotic cells, and decreased proliferation of thymocytes, which led to an immediate decrease in cellularity. FTOCs infected with vesicular stomatitis virus, known to act through TLR7, were similarly affected. Down-regulation of IL-7R α-chain expression, together with an increased expression of suppressor of cytokine signaling-1 and a concomitant decreased expression of the transcriptional regulator growth factor independence 1 were observed in TLR ligands or IFN-treated FTOCs. This indicates a role for these pathways in the observed changes in thymocyte development. Taken together, our data demonstrate that TLR activation and ensuing type I IFN production exert a deleterious effect on T cell development. Because TLR ligands are widely used as vaccine adjuvants, their immunomodulatory actions mediated mainly by IFN-α suggested by our results should be taken in consideration.

In Vitro Models of Human T Cell Development: Dishing Out Progenitor T Cells

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 sequen- tial, 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 ap- proach 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 treat- ment of immune-related diseases such as cancer, immunedeficiency, and autoimmunity. th -10 th week of gestation prevents the formation of the thymus and thus a complete absence of T cells (2, 3). Normal T cell development can be rescued in patients with DiGeorge syndrome following allogeneic transplantation of fetal thymic fragments (4, 5). The treatment of DiGeorge syndrome illustrates two important concepts: first, it estab- lishes the thymus as a unique microenvironment capable of sustaining T cell development without addressing the mo- lecular rationale for this dependency; and second, it estab- lishes that whole organ transplantation may be used success- fully to treat one rare form of primary T cell immunodefi- ciency in vivo provided this fetal tissue is available. Like the in vivo setting, the development of T cells in vitro has until recently also required donor thymic tissue. Recent insights into the key molecular players responsible for the thymic dependency of T cell development has permit- ted the differentiation of expanded hematopoietic progenitor