Differentiation patterns of CD4/CD8 thymocyte subsets in cocultures of fetal thymus and lymphohemopoietic cells from c-fos transgenic and normal mice.

This study examined the involvement of c-fos protooncogene in thymocyte development from lymphohemopoietic T cell progenitors, within the thymic microenvironment. We first analyzed the thymocytes developing in vitro in the fetal thymus from the c-fos transgenic mice and found a high proportion of CD4+ single positive (SP) cells. We then seeded either fetal liver or bone marrow (BM) cells from normal donors onto lymphocyte-depleted fetal thymus explants of c-fos transgenic mice. The results showed an increased proportion of mature CD4+ SP and decreased CD4+CD8+ double positive (DP) cells. A similar pattern of CD4/CD8 thymocyte subsets was observed when either thymus or BM cells from c-fos transgenic mice developed within a normal thymic stroma. The kinetics of thymocyte development in organ culture (from Days 3 to 11) suggested that the SP cells obtained under these conditions may have bypassed the CD4+CD8+ DP phase. It appears that the altered pattern of thymocyte development manifested in adult c-fos transgenic mice can be induced by the early embryonic thymic stroma, and may also involve cells in the lymphohemopoietic tissues.     

TCR signaling for initiation and completion of thymocyte positive selection has distinct requirements for ligand quality and presenting cell type

Thymocyte selection involves signaling by TCR engaging diverse self-peptide:MHC molecule ligands on various cell types in the cortex and medulla. Here we separately analyze early and late stages of selection to better understand how presenting cell type, ligand quality, and the timing of TCR signaling contribute to intrathymic differentiation. TCR transgenic CD4+CD8+ thymocytes (double positive (DP)) from MHC-deficient mice were stimulated using various presenting cells and ligands. The resulting CD69high cells were isolated and evaluated for maturation in reaggregate cultures with wild-type or MHC molecule-deficient thymic stroma with or without added hemopoietic dendritic cells (DC). Production of CD4+ T cells required TCR signaling in the reaggregates, indicating that transient recognition of self-ligands by DP is inadequate for full differentiation. DC bearing a potent agonist ligand could initiate positive selection, producing activated thymocytes that matured into agonist-responsive T cells in reaggregates lacking the same ligand. DC could also support the TCR signaling necessary for late maturation. These results argue that despite the negative role assigned to DC in past studies, neither the peptide:MHC molecule complexes present on DC nor any other signals provided by these cells stimulate only thymocyte death. These findings also indicate that unique epithelial ligands are not necessary for positive selection. They provide additional insight into the role of ligand quality in selection events and support the concept that following initiation of maturation from the DP state, persistent TCR signaling is characteristic of and perhaps required by T cells.     

Lck SH3 domain function is required for T-cell receptor signals regulating thymocyte development

Thymocyte development is shaped by signals from the T-cell antigen receptor. The strength of receptor signaling determines developmental progression as well as deletion of self-reactive T cells. Receptor stimulation of the extracellular signal-regulated kinase (ERK) pathway plays an important regulatory role during thymocyte development. However, it is unclear how differences in receptor signaling are translated into distinctive activation of the ERK pathway. We have investigated the potential role of the Lck tyrosine kinase in regulating intracellular signaling during thymocyte development. While Lck is known to be critical for initial T-cell receptor signaling events, it may have an independent role in regulating intracellular signaling through the function of its SH3 domain. To determine whether such a regulatory mechanism functions during thymocyte development, we generated mice in which the normal lck allele is replaced with an lck SH3 domain mutant. Analysis of these mice revealed that both early thymocyte development and maturation of CD4(+) and CD8(+) lineages is impaired. Investigation of thymocyte responses to antigen receptor stimulation showed a significant reduction in proliferation and ERK pathway activation, although initial signaling events were intact. These findings indicate that Lck SH3 domain function may provide a means to independently couple receptor signaling to regulation of the ERK pathway during thymocyte development.     

Finding their niche: chemokines directing cell migration in the thymus

T lymphocytes are generated throughout life, arising from bone marrow-derived progenitors that complete an essential developmental process in the thymus. Thymic T cell education leads to the generation of a self-restricted and largely self-tolerant peripheral T-cell pool and is facilitated by interactions with thymic stromal cells residing in distinct supportive niches. The signals governing thymocyte precursor migration into the thymus, directing thymocyte navigation through thymic microenvironments and mature T-cell egress into circulation were, until recently, largely unknown, but presumed to be mediated to a large extent by chemokine signalling. Recent studies have now uncovered various specific functions for members of the chemokine superfamily in the thymus. These studies have not only revealed distinct but also in some cases overlapping roles for several chemokine family members in various thymocyte migration events and have also shown that homing and positioning of other cells in the thymus, such as dendritic cells and natural killer T cells is also chemokine-dependent. Here, we discuss current understanding of the role of chemokines in the thymus and highlight key future avenues for investigation in this field.     

The Importance of LAT in the Activation,Homeostasis, and Regulatory Function of T Cells

LAT (linker for activation of T cells) is a transmembrane adaptor protein that plays an essential role in TCR-mediated signaling and thymocyte development. Because LAT-deficient mice have an early block in thymocyte development, we utilized an inducible system to delete LAT in primary T cells to study LAT function in T cell activation, homeostasis, and survival. Deletion of LAT caused primary T cells to become unresponsive to stimulation from the TCR and impaired T cell homeostatic proliferation and long term survival. Furthermore, deletion of LAT led to reduced expression of Foxp3, CTLA-4, and CD25 in T_reg cells and impaired their function. Consequently, mice with LAT deleted developed a lymphoproliferative syndrome similar to that in LATY136F mice, although less severe. Our data implicate that LAT has positive and negative roles in the regulation of mature T cells.     

Induction of thymocyte proliferation by supernatants from a mouse thymic epithelial cell line

The thymic stroma plays a critical role in the generation of T lymphocytes by direct cell-to-cell contacts as well as by secreting growth factors or hormones. The thymic epithelial cells, responsible for thymic hormone secretion, include morphologically and antigenically distinct subpopulations that may exert different roles in thymocyte maturation. The recent development of thymic epithelial cell lines provided an interesting model for studying thymic epithelial influences on T cell differentiation. Treating mouse thymocytes by supernatants from one of TEC line (IT-76M1), we observed an induction of thymocyte proliferation and an increase in the percentages of CD4-/CD8- thymocytes. This proliferation was largely inhibited when thymocytes were incubated with IT-76M1 supernatants together with an anti-thymulin monoclonal antibody, but could be enhanced by pretreating growing epithelial cells by triiodothyronine. We suggest that among the target cells for thymulin within the thymus, some putative precursors of early phenotype might be included.     

The role of dendritic cells in selection of classical and nonclassical CD8+ T cells in vivo

T cell development is determined by positive and negative selection events. An intriguing question is how signals through the TCR can induce thymocyte survival and maturation in some and programmed cell death in other thymocytes. This paradox can be explained by the hypothesis that different thymic cell types expressing self-MHC/peptide ligands mediate either positive or negative selection events. Using transgenic mice that express MHC class I (MHC-I) selectively on DC, we demonstrate a compartmentalization of thymic functions and reveal that DC induce CTL tolerance to MHC-I-positive hemopoietic targets in vivo. However, in normal and bone marrow chimeric mice, MHC-I+ DC are sufficient to positively select neither MHC-Ib (H2-M3)- nor MHC-Ia (H2-K)-restricted CD8+ T cells. Thus, thymic DC are specialized in tolerance induction, but cannot positively select the vast majority of MHC-I-restricted CD8+ T cells.     

A Cellular Automata-Based Mathematical Model for Thymocyte Development

Intrathymic T cell development is an important process necessary for the normal formation of cell-mediated immune responses. Importantly, such a process depends on interactions of developing thymocytes with cellular and extracellular elements of the thymic microenvironment. Additionally, it includes a series of oriented and tunely regulated migration events, ultimately allowing mature cells to cross endothelial barriers and leave the organ. Herein we built a cellular automata-based mathematical model for thymocyte migration and development. The rules comprised in this model take into account the main stages of thymocyte development, two-dimensional sections of the normal thymic microenvironmental network, as well as the chemokines involved in intrathymic cell migration. Parameters of our computer simulations with further adjusted to results derived from previous experimental data using sub-lethally irradiated mice, in which thymus recovery can be evaluated. The model fitted with the increasing numbers of each CD4/CD8-defined thymocyte subset. It was further validated since it fitted with the times of permanence experimentally ascertained in each CD4/CD8-defined differentiation stage. Importantly, correlations using the whole mean volume of young normal adult mice revealed that the numbers of cells generated in silico with the mathematical model fall within the range of total thymocyte numbers seen in these animals. Furthermore, simulations made with a human thymic epithelial network using the same mathematical model generated similar profiles for temporal evolution of thymocyte developmental stages. Lastly, we provided in silico evidence that the thymus architecture is important in the thymocyte development, since changes in the epithelial network result in different theoretical profiles for T cell development/migration. This model likely can be used to predict thymocyte evolution following therapeutic strategies designed for recovery of the thymus in diseases coursing with thymus involution, such as some primary immunodeficiencies, acute infections, and malnutrition.     

Positive selection of the T cell repertoire: where and when does it occur?

The T cell repertoire is shaped by both positive and negative influences. T lymphocytes that express the V beta 6 variable region are positively selected in the thymus by cells expressing major histocompatibility complex (MHC) class II E molecules. To identify these cells, we have quantitated V beta 6+ T lymphocytes in a set of transgenic mice showing variant patterns of E expression in the thymus. We demonstrate that class II molecules must be expressed on epithelial cells of the cortex for positive selection to occur. Using a direct assay of unmanipulated thymocytes, we show that positive selection is manifest only as a rather late event in thymocyte differentiation, after the maturation of cortical double-positives into single-positives.     

Expression of the p56lck Y505F Mutation in CD45-Deficient Mice Rescues Thymocyte Development

Mice deficient in the transmembrane protein tyrosine phosphatase CD45 exhibit a block in thymocyte development. To determine whether the block in thymocyte development was due to the inability to dephosphorylate the inhibitory phosphorylation site (Y505) in p56(lck) (Lck), we generated CD45-deficient mice that express transgenes for the Lck Y505F mutation and the DO11.10 T-cell antigen receptor (TCR). CD4 single-positive T cells developed and accumulated in the periphery. Treatment with antigen resulted in thymocyte apoptosis and the loss of transgenic-TCR-bearing cells. Peripheral CD45-deficient T cells from the mice expressing both transgenes responded to antigen by increasing CD69 expression, interleukin-2 production, and proliferation. These results indicate that thymocyte development requires the dephosphorylation of the inhibitory site in Lck by CD45.     

Early ontogeny of thymocytes in pigs: sequential colonization of the thymus by T cell progenitors

Successive colonization of the thymus by waves of thymocyte progenitors has been described in chicken-quail chimeras and suggested from studies in mice. In swine, we show that the first CD3epsilon-bearing thymocytes appear on day 40 of gestation (DG40). These early thymocytes were CD3epsilonhigh and belonged to the gammadelta T cell lineage. Mature CD3epsilonhigh alphabeta thymocytes were observed 15 days later (DG55), and their occurrence was preceded by the appearance of CD3epsilonlow thymocytes (DG45). Thereafter, we observed transient changes in thymocyte subset composition (DG56-DG74), which can be explained by a gap in pro-T cell delivery to the thymus. This delivery gap corresponds with the expression of the pan-leukocyte CD45 and pan-myelomonocytic SWC3a markers in fetal liver and bone marrow and is probably caused by shifting of primary lymphopoiesis between these organs. Therefore, we conclude that the embryonic thymus is colonized by at least two successive waves of hemopoietic progenitors during embryogenesis and that the influx of thymocyte progenitors is discontinuous. Surface immunophenotyping and cell cycle analysis of thymocyte subsets allowed us to compare thymocyte differentiation in pigs with that described for rodents and humans and to propose a model for T cell lymphopoiesis in swine. We also observed that the porcine IL-2Ralpha (CD25), a typical differentiation marker of pre-T cells in mice and humans, was not expressed on thymocyte precursors in pigs and could only be found on mature thymocytes. Finally, we observed a subset of TCRgammadelta+ thymocytes that were cycling late during their development in the thymus.     

Regulatory T cells in the establishment and maintenance of self-tolerance: role of the thymic epithelium

The thymus constitutes the microenvironment for T lymphocyte differentiation and acquisition of self-tolerance. Aiming to specify the contributions of the two essential parts of the thymus, namely hemopoietic and epithelial, we have devised experimental models in birds and mice. Chimeric thymuses, xenogeneic in birds and allogeneic in mice, were constructed early in development. In both models we could demonstrate a critical role of the epithelial component of the thymic stroma in induction and maintenance of self-tolerance. These experiments showed that suppression mechanisms are also implicated in these events, strongly suggesting the existence of regulatory T cells in both models. Before these experiments the control of self-tolerance was usually attributed to suppressive cells. However, as the cell phenotypes were not identified, the role of these cells was disregarded. Numerous studies since our investigations argue in favour of regulatory mechanisms. The work we initiated several years ago represents a contribution to our understanding of the two linked and opposite aspects of immune-responded control, namely self-tolerance and autoimmunity.