Lisa M. Spain

Distinct roles of prostaglandin H synthases 1 and 2 in T-cell development

Prostaglandin G and H synthases, or cyclooxygenases (COXs), catalyze the formation of prostaglandins (PGs). Whereas COX-1 is diffusely expressed in lymphoid cells in embryonic day 15.5 thymus, COX-2 expression is sparse, apparently limited to stromal cells. By contrast, COX-2 is predominant in a subset of medullary stromal cells in three- to five-week-old mice. The isozymes also differ in their contributions to lymphocyte development. Thus, experiments with selective COX-1 inhibitors in thymic lobes from normal and recombinase-activating gene-1 knockout mice support a role for this isoform in the transition from CD4(-)CD8(-) double-negative (DN) to CD4(+)CD8(+) double-positive (DP). Concordant data were obtained in COX-1 knockouts. Pharmacological inhibition and genetic deletion of COX-2, by contrast, support its role during early thymocyte proliferation and differentiation and, later, during maturation of the CD4 helper T-cell lineage. PGE2, but not other PGs, can rescue the effects of inhibition of either isoform, although it acts through distinct EP receptor subtypes. COX-dependent PG generation may represent a mechanism of thymic stromal support for T-cell development.

Altered aging-related thymic involution in T cell receptor transgenic, MHC-deficient, and CD4-deficient mice.

During aging in mice and humans, a gradual decline in thymus integrity and function occurs (thymic involution). To determine whether T cell reactivity or development affects thymic involution, we compared the thymic phenotype in old (12 months) and young (2 months) mice transgenic for rearranged alphabeta or beta 2B4 T cell receptor (TCR) genes, mice made deficient for CD4 by gene targetting (CD4(-/-)), mice made deficient for major histocompatibility complex (MHC) class I (beta2M-/-) or class II genes (A(beta)(b-/-) on C57Bl/6 background) or both. The expected aging-related reductions in thymic weights were observed for all strains except those bearing disruption of both class I and class II MHC genes. Therefore, disruption of MHC class I and class II appeared to reverse or delay aging-related thymic atrophy at 12 months. Immunohistochemical analysis of aging-associated alterations in thymic morphology revealed that TCR alphabeta transgenes, CD4 disruption, and MHC class II disruption all reduced or eliminated these changes. All strains examined at 12 months showed alterations in the distribution of immature thymocyte populations relative to young controls. These results show that aging-associated thymic structural alterations, size reductions, and thymocyte developmental delays can be separated and are therefore causally unrelated. Furthermore, these results suggest that the T cell repertoire and/or its development play a role in aging-related thymic involution.

Quantitative Analysis of the Efficiency of Clonal Deletion in the Thymus

One of the major mechanisms for establishing self-tolerance is the clonal deletion of self-reactive T cells during their development in the thymus. Using a TCR transgenic mouse model, we have established a quantitative ex vivo assay for examining the sensitivity and specificity of negative selection. Thymic organ cultures established from mice of varying MHC haplotypes were incubated with antigen, and the efficiency of clonal deletion assessed. We show here that clonal deletion of CD4+8+ thymocytes is sensitive to both the gene dosage and the allelic variation of MHC class II molecules expressed on thymic antigen-presenting cells. We also find that when epithelial cells in the thymic cortex are the only antigen-presenting cells expressing the appropriate MHC class II molecules, negative selection of CD4+8+ cells is as efficient as when antigen is presented on all thymic antigen-presenting cells. These studies demonstrate that the induction of self-tolerance via clonal deletion in the thymus is a function not only of antigen concentration, but also of MHC class II cell-surface density. In addition, together with the reports of others, these results confirm that cortical epithelial cells can mediate negative selection, and demonstrate that they do so in the intact thymic microenvironment.

Fetal thymic organ culture in rotating bioreactors

Dear Editor: The task of the thymus is to support the differentiation of T cells whose T cell receptors (TCRs) interact with major histocompatibility complex (MHC) proteins (positive selection), and to eliminate by deletion any T cells bearing autoreactive TCRs (negative selection). Our understanding of the mechanism of thymic function has been greatly enhanced by the development of a technique for in vitro culture of intact thymus, so-called fetal thymic organ cultures (reviewed in 17). The use of fetal thymie organ cultures has established the role of peptides in negative and positive selection (reviewed in 15), the role of signaling pathways in T cell development (1,10), the cellular basis of T cell development and selection (2,23) and the enumeration and characterization of T cell progenitors (20,27,28,31,32). Thymic organ culture also has clinical application in the removal of resident thymocytes before thymic organ transplantation for DiGeorge anomaly patients (19). Despite these successful applications of fetal thymic organ culture technology, development under standard culture conditions is not optimal, as evidenced by low cellular recoveries, the appearance of cell types not seen in vivo, and skewing of the normal subset ratios (5,26). These effects could be due in part to changes in thymic mieroarehitecture associated with ex vivo euhuring. For example, the thymie lobes become flattened, and stromal cells adhere to the culture substratum and grow out of the organ. In addition, in vitro cultured thymus does not have access to the normal influx of progenitors. In summary, both experimental and clinical applications require that new strategies for thymic culturing be explored. The experiments described here have multiple goals. First, we have investigated the ability of rotating wall bioreaetors to support T cell development in thymie organ euhure. In addition, we have investigated whether gravity vector randomization, as occurs in the rotating bioreactor, has any effect on the kinetics and extent of T cell development in thymus explants. Our first experiments were designed to test the viability of fetal thymus explants in bioreaetor vessels. We used fetal thymic lobes from embryonic Day 17 (El7) B10.BR mice, at which stage the majority of thymocytes are undergoing positive and negative selection but are beyond the major stage of proliferation. We used standard liquid medium (17) and cultured the explants in a rotating bioreactor (16 rpm) or on sterile sponges floating on medium in dishes (standard air-medium interface conditions) (17). Because it has been previously shown that TCR ct~3 thymoeyte development requires 40-80% oxygen levels (29), we preeharged the medium intended for the bioreactor and continuously supplied the bioreactor with 95/5% O2/CO2 gas mixture. After 6 d of culture the lobes were removed and dissociated by mechanical disruption, and the cells were counted and stained for T cell developmental markers. T cell progenitors are negative for both CD4 and CD8 (CD4 8-) as they begin rearrangement of their TCR genes. Once TCR rearrangement at the TCR ~3 locus is productive, the progenitors upregulate CD8 and shortly thereafter becmne CD4+8 +. At this stage, rearrangement at the TCR c~ locus is completed and positive and negative selection based on the TCR specificity is underway. The CD4+8 + thymocytes are short-lived: they die by programmed cell death if they do not receive a positive selection signal. Following selection, T cells differentiate into one of the two mature subsets, which express only one of the two coreceptor molecules (helper CD4 + or cytotoxic CD8+). Therefore, to assess development we stained the cells with fluorescently-labeled antibodies against the CD4 and CD8 coreceptors and analyzed the stained ceils using flow cytometry. This analysis showed that the bioreactor conditions were not toxic to preformed E17 thymocytes, because numbers ofCD4+8 + thymocytes recovered from the rotating bioreactor and standard air-medium interface conditions were similar (data not shown). We next compared the development of thymocytes beginning from El5, when proliferation was active and thymocytes were mainly CD4 8 precursors. El5 fetal thymic lobes were cultured in the bioreactor in liquid medium as described above. However, in this case, one lobe from each thymus was placed in identical bioreactors, one of which was kept stationary while the other rotated at 16 rpm. After 6 d, the cultures were terminated and analyzed for coreceptor expression by flow cytometry as described above. We observed that each lobe could be categorized with respect to its development, based on the following criteria: cellular recovery, percentage of mature CD4 + thymocytes (CD8 thymocytes could not be used because some are immature), and ratio of CD4+8 + to CD4 8thymocytes. We scored as fully developed any lobes that contained more than 104 cells and more than 50% CD4+8 + and 5% CD4 + thymocytes; partially developed, any lobe that contained more than 40% CD4+8 + but few C D4+; and undeveloped, any lobes that contained fewer than 103 cells with less than 5% CD4+8 + thymocytes. Under these liquid medium conditions, we found that 46% of thymic lobes were undeveloped in the rotating compared to none in the stationary condition, while 89% of lobes were fully developed in the stationary bioreactor compared to only 18% in the rotating condition (Table 1). In addition to categorizing the development of each lobe, we further analyzed the lobes that did show development. The percentages of cells in