By the mid-1970s, immunologists had made it clear that there was a real division of labor with regard to the role played by lymphocytes in immune responses. Importantly, lymphocytes were known to be phenotypically and functionally heterogeneous and to be produced at distinct anatomic sites. Cells capable of producing Abs had been shown to be generated in the bursa of Fabricius in birds (1) and in the fetal liver of mice (2), whereas the thymus had been shown to be a key immunological site (3) that fostered the production of lymphocytes with cytotoxic effector function (4). These observations, together with seminal thymectomy/bursectomy studies by Cooper and colleagues in the chicken (5), clearly showed that the thymus and the bursa/fetal liver played distinct roles in the development of lymphocyte subsets. Importantly, antisera were also available by this time that allowed the identification of these key lymphocyte subsets, with markers such as surface Ig and θC3H (now termed Thy-1 or CD90) being used to identify Ab-producing cells and thymus-derived cells, respectively (4, 6, 7). Despite these advances, experimental systems were required that provided the manipulability to dissect and study the developmental requirements of these distinct lymphoid lineages. Indeed, two years earlier Owen and colleagues used an organ culture system showing that the mouse fetal liver represented a mammalian bursa equivalent, providing an accessible in vitro system to study B cell development in the mouse (2). With regard to T cells, although other studies had reported use of thymus organ culture in the chick (8), there was a clear need for a system that generated T cells in the mouse, a species emerging as a key model of choice for many immunologists. Through the use of mouse fetal thymus organ culture (FTOC)2 techniques, Robinson and Owen (9) showed that functionally competent T cells could be generated within explanted thymic tissue under controlled conditions in vitro, paving the way for the study of mechanisms regulating T cell development.

By adopting approaches that had previously been described and used to study embryonic organ development, including thymus (10, 11, 12), Robinson and Owen described a method in which embryonic mice at embryonic days 14 and 15 of gestation were used as a source of thymus lobes, which at this developmental stage contained only large blast-like cells thought to represent lymphocyte precursors. After explantation into organ culture on the surface of floating membrane filters, thymic cultures were shown to undergo significant increases in size, correlating with an increase in lymphocyte recovery of up to 14-fold by day 7 of culture. Such culture conditions were also shown to support the emergence of lymphocytes with a size typical of peripheral lymphocytes, as well as the acquisition of the surface marker Thy-1. Importantly, B cells were found to be absent from thymus organ cultures, an important indication that the specialization of the thymus for the production of T cells in vivo could also be recapitulated in vitro. Perhaps most significantly, Robinson and Owen also treated lymphocytes generated in FTOC with the T cell mitogens PHA and Con A, together with 125I-labeled iododeoxyuridine. This experiment was important for two reasons. Firstly, it showed that functionally competent T cells capable of undergoing proliferation in response to stimulation could be generated in vitro in the context of the thymic microenvironment. Secondly, it also showed the ease with which the FTOCs and the T cells generated within them could be manipulated by the addition of exogenous reagents that could enable the study of stages in T cell development. For example, the authors went on to show that FTOC also generated T cells that responded to alloantigens in mixed leukocyte culture (13) and that tolerance to alloantigens could be induced by coculturing thymus with fragments of histoincompatible spleen (14).

Since its initial description, the FTOC system has been used extensively to study the mechanisms regulating key events that occur during intrathymic T cell development. For example, FTOC proved to be a key system in unraveling the role of MHC-bound peptides in the positive and negative selection of the developing TCR repertoire by enabling study of the effects of individual peptides on a relatively synchronous wave of thymocyte development in the absence of effects on peripheral T cells (15, 16). In addition, the “standard operating procedure” for FTOC has undergone several revisions that have allowed study of the role of the thymic microenvironment in T cell production, including thymic epithelial cell development and function. The ability to generate alymphoid FTOCs by exposure to 2-deoxyguanosine (17) and the production of reaggregate thymus organ cultures from defined stromal components (18) are notable in this context, the latter currently being used as a model to study thymocyte migration and development in real time using two-photon microscopy (19).

In summary, the description by Robinson and Owen (9) of an in vitro system to study T cell development has proved to be a cornerstone for our understanding of thymic function and the stages and events that occur during T cell development. That this system still remains the only in vitro system capable of supporting a full program of T cell development, from progenitor recruitment to MHC restriction and clonal deletion is a testament to the use of simple experimental approaches to study complex biological events.

The authors have no financial conflict of interest.

2

Abbreviation used in this paper: FTOC, fetal thymus organ culture.

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