Reaggregate Thymic Organ Cultures Research Articles

The in vivo development of human T cells from CD34(+) cells in the murine thymic environment.

There is increasing evidence that human hematopoietic stem cells can develop into lymphocytes expressing T cell surface markers in the organ culture of murine embryonic thymic lobes. If human T cells with functional maturity are inducible from human stem cells in the mouse, it may be a useful model to investigate human T cell development and the human immune response in vivo. To approach this, we produced a hybrid cluster of murine fetal thymic epithelial cells and human cord blood-derived CD34(+) cells (hu/m cluster) using reaggregate thymic organ culture, and subsequently implanted it under the kidney capsule of NOD/SCID mice. The implanted hu/m cluster grew in volume under the kidney capsule and contained increased numbers of CD4(+)CD8(+)cells as well as CD4 or CD8 single-positive cells with low CD1a expression. These lymphocytes were also shown to possess activity for producing IL-2 and IL-4. Characteristics similar to human T cells also developed in the thymus of newly established mice lacking NK activity from NOD/SCID mice. These results indicate that functionally mature T cells can develop in vivo from human hematopoietic progenitors in the murine environment composed of thymic epithelial cells.

Book Review

Haematopoietic and lymphoid cell culture. Margaret J. Dalman and Jonathan R. Lamb. Cambridge University Press, 2000. Pp. 188. £55·00. ISBN: 0-521-62969-1 (paperback). This is a most succinct and clearly written handbook describing the characteristics and methods of isolation and culture of the major types of haematopoietic cells. The cell types discussed are embryonic stem cells, dendritic cells, murine thymic explant cultures, T cells, B lymphocytes, monocytes and macrophages, NK and LAK cells, and, lastly, mast cells and basophils. An obvious omission, however, are multipotent haematopoietic stem cells and their progenitors. Each chapter is devoted to each of the above cell types and written by appropriate experts in the field. The chapters are well laid out and structured, each with a clear and comprehensive introduction summarizing the origin, localization, development, phenotypic features, culture requirements and functions of each cell type. This is followed by a detailed and practical summary of methodologies for their isolation and in vitro culture. The book includes helpful explanatory diagrams and well-presented photographs of the various cells types grown in culture. The chapter on embryonic stem cells describes the conditions for growth, aggregation and differentiation of embryonic stem cells and analysis of differentiated progeny. For dendritic cells, their generation in culture from mouse bone marrow in human peripheral blood monocytes is discussed, as well as an assessment of the migratory properties. The chapter on thymic explant cultures discusses the culture of murine, fetal and newborn thymic organ, and the reaggregate thymus organ cultures, to assess the interaction of isolated lymphoid cells and thymic stromal cells as an in vitro model of the thymus. The chapter on T cells discusses the isolation and expansion of T cell lines and clones. For B lymphocytes, the culture of early bone marrow-derived B cells, and mature B lymphocytes from spleen and the peritoneal cavity in human and mouse, are described. In addition, flow cytometry and molecular techniques for phenotypic analysis are included. The chapter on monocytes and macrophages describes the isolation and culture of these cells from a variety of tissues in mouse and human, and the chapter on NK and LAK cells describes the identification, culture and cloning of murine and human NK cells. Finally, the culture of mast cells and basophils in mouse and human is detailed, together with the growth of mast cells from human CD34+ progenitor cells. Protocols for the detection of these two cell types are also included. In conclusion, this handbook is an invaluable and easily readable reference. Its attractively compact size makes it easily accessible not only on book shelves, but it will also slip easily in to briefcases and laboratory coat pockets. It is just the type of eagerly sought after book that can easily sprout legs and wander off into neighbouring offices and pockets!

A bone marrow-derived stroma cell line, ST2, can support the differentiation of fetal thymocytes from the CD4+ CD8+ double negative to the CD4+ CD8+ double positive differentiation stage in vitro.

T-cell precursors differentiate into mature T cells predominantly in the thymus. However, it has also been reported that T-cell precursors mature in extrathymic organs such as the liver, bone marrow, or intestines. In order to investigate the nature of the extrathymic microenvironment that supports T-cell maturation, we examined the effect of a bone marrow-derived stroma cell line, ST2, on T-cell precursors by using a reaggregate thymic organ culture (RTOC) system. We found that ST2 cells supported the differentiation of fetal thymocytes at day 14.5 of gestation from a CD4- CD8- double negative (DN) to a CD4+ CD8+ double positive (DP) differentiation stage in a manner similar to that observed in thymus. Anti-interleukin-7 receptor (IL-7R) and anti-c-kit antibodies blocked the growth of thymocytes in RTOC with ST2 cells, but did not inhibit the generation of DP thymocytes. These data indicate that a bone marrow-derived stroma cell, ST2, which supports B-cell differentiation, is also able to support T-cell development and may constitute one of the microenvironmental components for extrathymic T-cell development.

In vitromodels of T cell development

A number ofin vitrosystems are currently being used to study both thymocyte development and thymic stromal cell function. However, the usefulness of dispersed culture systems is limited since they often involve disruption of interactions within the normal three-dimensional architecture of the thymusin vivowhich are critical for normal development to proceed. In contrast, Fetal Thymus Organ Culture (FTOC) provides an experimental system where such interactions are maintained, thereby allowingin vitroaccess to key aspects of thymocyte development. More recently, Reaggregate Thymus Organ Cultures (RTOCs) have allowed detailed analysis of thymic stromal cell function, while retroviral transfection of thymocyte subsets under FTOC conditions provides a rapid means to investigate thymocyte development at the molecular level. Current use of the FTOC approach is summarised here, and where appropriate is compared to the use of dispersed culture systems.

Differentiation of CD4(high)CD8(low) coreceptor-skewed thymocytes into mature CD8 single-positive cells independent of MHC class I recognition.

Thymocytes with a CD4(hi)CD8(lo) coreceptor-skewed (CRS) phenotype have been shown to contain precursors for CD8 single-positive (SP) thymocytes, in addition to precursors for CD4 SP cells. The selection mechanisms that stimulate CD4(hi)CD8(lo) cells to revert to the CD8 lineage are not known. Mice transgenic (tg) for the major histocompatibility complex (MHC) class I-restricted P14 T cell receptor (TCR), on the H-2bm13 background, generate a large number of CD4(hi)CD8(lo) CRS thymocytes. We analyzed the developmental potential and the differentiation requirements of the CD4(hi)CD8(lo) population of these mice. Using reaggregate thymic organ cultures (RTOC), we observed that these cells efficiently and almost exclusively differentiate into CD8 SP cells. Differentiation occurred independent of whether or not the MHC haplotype of the thymic stroma corresponds to the MHC restriction of the tg TCR. Loss of CD4 was independent of thymic stroma, up-regulation of CD8 to full levels was dependent on thymic stroma but independent of MHC haplotype. After trypsin treatment and overnight incubation, these CRS cells re-expressed CD8 but failed to re-express CD4, indicating that they are in the process of terminating CD4 synthesis. CD8 SP cells derived from the CRS cells proliferate in response to peptide-pulsed antigen-presenting cells. Our data suggest that CD4(hi)CD8(lo) CRS thymocytes bearing the P14 tg TCR have completed positive selection and differentiate autonomously into functionally competent CD8 SP cells.

A novel mouse thymocyte antigen (F3Ag): down-regulation during the CD4+CD8+ double-positive stage indicates positive selection.

We describe a novel mAb (F3) which reacts with a 65 kDa thymocyte surface protein, expressed on approximately 80% of thymocytes, referred to as F3Ag. In ontogeny, F3Ag expression begins in the CD4(-)CD8(-) double-negative (DN) CD25(+) population and is maintained through approximately 85% of the CD4(+)CD8(+) double-positive (DP) stage. DP cells with high TCR expression and CD4(+) single-positive (SP) cells are predominantly negative for F3Ag, whereas many CD8(+) SP thymocytes express F3Ag. F3Ag-DP thymocytes show a reduced expression of RAG-1 and RAG-2 compared with F3Ag+ DP cells. The shutdown of F3Ag expression during the DP stage is related to positive selection: mice deficient for MHC class I and class II molecules maintain F3Ag expression in almost all DP cells. Transgenic (tg) mice carrying TCR restricted for MHC class II show a more pronounced down-regulation of F3Ag in the DP compartment than normal mice, depending on the presence of a positively selecting MHC. The size of the F3Ag- DP subset is positively correlated with the efficacy of positive selection into the CD4(+) SP compartment. Because some CD8(+) SP cells express F3Ag, the relationship between F3Ag down-regulation and positive selection is less obvious in DP cells of mice carrying MHC class I-restricted tg TCR. However, in reaggregate thymic organ cultures, sorted F3Ag- DP cells differentiate into CD8(+) SP cells more rapidly than do F3Ag+ DP cells. Thus, after down-regulation in the DP stage, a proportion of CD8(+) SP cells appears to re-express F3Ag. In addition, the proportion of F3Ag-CD8(+) SP cells depends on the efficacy of positive selection into the CD8 lineage. Taken together, the regulation of the expression of the F3Ag appears to be associated with signals that control thymic repertoire selection.