Hematopoietic development and immunological tolerance

The immune system can discriminate between self and non-self, thus assuring clearance of infections by both innate and adaptive effector cells without auto-aggression, thus, establishing tolerance. Projects discussed within this thesis focus on hematopoiesis with special emphasis on T cell development and acquisition of self-tolerance.
A multitude of signals derived from cell-cell interactions and/or cytokine signaling determine the first developmental steps of HSCÕs differentiating towards the lymphoid lineage. Instructive cytokine signaling that drives cells into differentiation towards the next progenitor stage or a specified cell type by actively switching genes on or off is rarely known. Our data from project I, introduces FLT3L as a cytokine with such an effect. In vivo administration of FLT3L promotes expansion of progenitors with combined myeloid and lymphoid potential. To investigate further the role of FLT3L in hematopoietic development, we generated transgenic mice expressing high levels of human FLT3L. FLT3L transgenic mice displayed a dramatic expansion of dendritic and myeloid cells, leading to splenomegaly and blood leukocytosis. Bone marrow myeloid and lymphoid progenitors were significantly increased in numbers but retained their developmental potential. Furthermore, transgenic mice developed anemia together with a reduction in platelet numbers. FLT3L was shown to rapidly reduce erythrocyte numbers when injected into wild-type mice, indicating a direct negative role of the cytokine on erythropoiesis. We conclude that FLT3L acts on multipotent progenitors in an instructive way, inducing their development into myeloid/lymphoid lineages while suppressing their megakaryocyte/erythrocyte potential.
The aim of project II was to set up an in vitro T cell culture system which is not based on OP9-DL1 or OP9-DL4 stromal cell lines for studying early T cell development prior to T cell selection. Notch-ligand expression by OP9 stromal cells may vary; thus, reproducibility of culture conditions has been proven difficult. To address timing, durability and reversibility of Notch signaling effects on hematopoietic precursors, the Òplastic thymusÓ in vitro culture system was developed. To provide precursors with an adequate signal, a DLL4-Fc fusion protein is immobilized at the surface of cell culture flasks. The Fc-part of the fusion molecule is fixed to the surface by means of a monoclonal IgG anti-Fc antibody, thus directing the orientation of DLL4 towards the cells. We have studied the ability of surface-bound DLL4-Fc to induce prolonged and extensive expansion of ex vivo isolated Sca1+ c-kit+ fetal liver or adult BM haematopoietic progentiors (LSKÕs). Functionality of in vitro generated pro T cells could be demonstrated by transplantation experiments. ÒPlastic thymusÓ derived pro T cells could be shown to reconstitute the thymus of irradiated CD3-/-, preTalpha-/- or WT mice and generate a functional peripheral T cell compartment of both CD8+ and CD4+ antigen reactive T cells.
Autoimmune diseases develop when self-specific T cells that escaped negative selection initiate a harmful immune response against self. In project III, by establishing a double transgenic mouse system in which different amounts of a cell-surface neo-self antigen are expressed under the CD11c promoter, we demonstrate that antigen dose dramatically influences T cell tolerance mechanisms. Moderate antigen expression favors the development of antigen-specific regulatory T cells and the establishment of a tolerogenic environment. In marked contrast, high dose of antigen expression results in very stringent negative selection, in poor development of antigen-specific regulatory T cells and the early onset of anemia and splenomegaly and the late development of arthritis and high titers of IgG auto-antibodies. Disease is initiated by autoreactive T cells, which escape negative selection by expressing a second T cell receptor with a different specificity or an altered affinity. Transfer of antigen-specific regulatory T cells ameliorates the early onset signs of disease but does not prevent the development of long-term chronic pathologies.
Some signs of potential autoimmunity, such as the presence of anti-nuclear antibodies (ANA), become more prevalent with age. In most cases, these elderly people remain healthy. In project IV we have investigated whether the same holds true for inbred strains of mice. We found, that when they reach the age of 12 months, almost all mice of the C57BL/6 (B6) strain spontaneously produce high titers of IgG ANA. At this time, large numbers of germinal centers are present in the spleen, IgG deposition can be found in kidney glomeruli and lymphocyte infiltrates are found in the salivary glands. Despite all these signs of a potential autoimmune response, the mice remain healthy. In contrast to B6 mice, DBA/2 mice do not produce IgG ANAÕs at that age. The F1 hybrids of these two strains (BDF1), show an intermediate incidence and lower titers of IgG ANA, pointing out the importance of genetic background and suggesting a mechanism of suppression of ANA production in DBA/2 mice. The production of ANA is CD4 T cell dependent, since B6 mice deficient for MHC class II do not produce IgG ANAÕs upon ageing. Experiments with irradiated bone marrow chimeras clearly show that ANA production is not determined by age related changes in radiosensitive, hematopoietic precursor cells, and that the CD4 T cells that promote ANA production is radioresistent. Thymectomy of young adult B6 mice leads to premature alterations in T cell homeostasis and ANA production, mimicking the conditions observed in old mice. Our findings indicate that a disturbed T cell homeostasis is closely associated with and likely to drive the onset of some autoimmune features.