Synthetic Immunology by Takeshi Watanabe & Yousuke Takahama

Author:Takeshi Watanabe & Yousuke Takahama
Language: eng
Format: epub
Publisher: Springer Japan, Tokyo

T Cell Development in Vitro

Use of Ex Vivo Tissue Fetal Thymic Organ Cultures (FTOC)

Thymus explants from embryonic day 14–15 mice, also known as fetal thymic organ cultures (FTOC), were the earliest material used for differentiation of HSCs into T lineage cells in culture (Jenkinson et al. 1982). Treatment with purine nucleoside, 2-deoxyguonosine, is selectively toxic to lymphocytes, depleting the T cells, while leaving thymic stromal architecture intact. After washing out the 2-deoxyguanosine, hematopoietic progenitor cells (e.g. derived from day 15 fetal liver or adult bone marrow) from another mouse, possibly with a different genotype, can be introduced into these “empty” FTOCs, which are still able to support T lymphopoiesis (Jenkinson and Owen 1990). This was the first indication that the T cell generation could be replicated in vitro.

The progenitor cells seeding the FTOC go through the normal ontogeny of T cell development as they would in the fully formed thymus. In their early stages of development, the thymocyte progenitors are referred to as double negative cells (DNs) as they still lack both CD4 and CD8 expression. The hallmark of the developing DNs is the sequential acquisition and loss of CD25 and CD44 expression. Many different molecular cues combine at each different substage of DN development to allow progression from DN1 (CD44+ CD25-) to DN2 (CD44+ CD25+), DN3 (CD44- CD25+) and DN4 (CD44- CD25-) before acquiring the expression of both CD8 and CD4 markers at the double positive (DP) stage (Godfrey et al. 1993). Normally, a fully formed thymus contains approximately 3 % DNs, 85 % DPs, 4 % CD8+ single positive (SP) and 8 % CD4+ SPs. Many of the cellular and molecular signals required to advance through T cell development were initially deciphered thanks to the use of FTOCs (Anderson and Jenkinson 2008).

This chimeric system offered an in vitro approach that facilitated many advances for describing the role of TECs versus the hematopoietic cell component during development. Genetic mutations or gene knockouts (KO) cause aberrations in T cell development. In many cases, it is not clear whether the cause of deficiency is attributable to the hematopoietic or/and the stromal component. To verify which component was responsible for the aberration, in vivo adoptive transfer method are typically used. HSCs from the KO or mutant mouse are transplanted into a wild-type (WT) host, irradiated to deplete its own hematopoietic cells, followed by the characterization of the previously described developmental checkpoints. Together with the reciprocal experiment using WT HSC transplants into mutant recipients, the gene of interest’s role in thymic or hematopoietic contribution can be determined. Nonetheless, FTOCs provide a faster, more direct and less expensive way to examine the same question; i.e. by transferring WT HSCs into depleted mutant FTOCs and mutant HSCs into WT FTOCs (Anderson and Jenkinson 2008).

In addition, FTOCs provided a more accessible and specific method of studying T cell development. For example it was now possible to study lineage fate choices of a single progenitor cell as it could now be injected into the FTOC (Watanabe et al.

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