Lymphoid progenitors from bone marrow enter and move through the thymus where they are exposed to an array of signals responsible for their progression through T cell lineage pathways.1,2 The potential complexity generated by the many possible combinations of signals led to the notion that simple in vitro culture systems would not be sufficient to support the intricacies of T cell development. Until recently, models of T cell differentiation were limited to more complex systems including fetal thymic organ cultures (FTOCs) designed to more closely mimic the natural environment.3 Although FTOCs are able to support T cell differentiation, their relative complexity compared to 2 dimensional cultures makes it more difficult to tease out the mechanisms controlling T cell development.4 Recently, advances have been made with simplified in vitro systems that may lead to a greater understanding of the major players associated with the basic elements of T cell differentiation.5,6
|Figure 1. Embryonic stem cells (ESCs) cells were cultured on a monolayer of OP9 cells expressing the Notch ligand, Delta-like 1 (OP9-DL1). Following the subsequent addition of Flt-3 Ligand and IL-7, and
several passages over a period of approximately three weeks, ESCs differentiate into several T cell subsets.
Part of the mechanism known to be critical for directing T cell fate occurs via the Notch signaling pathway.7 Notch is evolutionarily conserved, regulating cell fate decisions in a number of cell and tissue types.8 Ligand binding by members of the Jagged or Delta-like families results in the proteolytic cleavage and release of the intracellular fragment of the Notch heterodimer.9 Translocation to the nucleus then allows for its regulation of gene expression. Notch-1, specifically, has been shown to be critical for the establishment of T cell fate. Loss of function results in the blockade of T cell development and enhanced B cell production, while over-expression results in the blockade of B cell lymphopoiesis and leads to the generation of T cells.10-12
Embryonic stem cells (ESCs) co-cultured on a monolayer of the bone marrow stromal cell line OP9 exhibit the ability to differentiate into most hematopoietic lineages, except for T cells.13,14 Schmitt et al. demonstrate the in vitro conditions necessary to drive totipotent ESCs through T cell differentiation.5 Given the known importance of Notch signaling in T cell development, OP-9 cells were transfected with the Notch ligand Delta-like 1 (OP9-DL1) and co-cultured with ESCs. Regular passages of non-adherent cells and supplementation with Flt-3 Ligand and IL-7 over a period of approximately 3 weeks resulted in the generation of several T cell subsets including ?d+, aß+CD4+CD8+, and αβ+CD4-CD8+ cells (Figure 1). In response to antigen, mature CD4-CD8+ T cells isolated from these cultures proliferated and generated an IFN-γ response. Mature CD4+ helper T cells were missing using this technique, probably as a result of blocked development due to limited MHCII expression by OP9 cells.5 However, if T cell progenitors were isolated from OP9-DL1 co-cultures and transferred to fetal thymic lobes, the entire T cell repertoire could be reconstituted in vivo when the lobes were transplanted into immunodeficient mice. Immunodeficient mice with transplants also exhibited the ability to generate an effective viral immune response. Because of the relatively easy access to developing lymphoid cells in OP9 co-cultures and the ability to genetically manipulate ESCs, this simplified system will undoubtedly advance the understanding of fundamental mechanisms underlying T cell differentiation. In addition, techniques such as these could provide a renewable source of T lymphocytes with therapeutic potential for a variety of immune disorders.
- Lind, E.F. et al. (2001) J. Exp. Med. 194:127.
- Bommhardt, U. et al. (2004) Cell. Mol. Life. Sci. 61:263.
- Kamarck, M.E. & P.D. Gottlieb (1977) J. Immunol. 119:407.
- Jenkinson, E.J. & G. Anderson (1994) Curr. Opin. Immunol. 6:293.
- Schmitt, T.M. et al. (2004) Nat. Immunol. 5:410.
- de Pooter, R.F. et al. (2003) Blood 102:1649.
- Radtke, F. et al. (2004) Curr. Opin. Immunol. 16:174.
- Harper, J.A. et al. (2003) Clin. Genet. 64:461.
- Schweisguth, F. (2004) Curr Biol. 14:R129.
- Han, H. et al. (2002) Int. Immunol. 14:637.
- Wilson, A. et al. (2001) J. Exp. Med. 194:1003.
- Pui, J.C. et al. (1999) Immunity 11:299.
- Nakano, T. et al. (1994) Science 265:1098.
- Cho, S.K. et al. (1999) Proc. Natl. Acad. Sci. USA 96:9797.