G. Dorban

Aire and Foxp3 Expression in a Particular Microenvironment for T Cell Differentiation

Objective: The thymus is the primary lymphoid organ responsible for T cell development and the establishment of central self-tolerance. Among thymic epithelial cells, thymic nurse cells (TNC) interact closely with immature thymocytes and constitute a special microenvironment for T cell differentiation and selection. In addition, TNC express neuroendocrine self-antigens such as oxytocin and insulin-like growth factor-2, whose intrathymic transcription is regulated by the autoimmune regulator gene/protein (Aire). Both effector and natural regulatory T cell (nTreg) lineages develop in the thymus, but the mechanisms leading to nTreg selection in the thymus are still unclear. Foxp3 is the most specific nTreg marker that is required for nTreg functional activity, but not for engagement into the Treg lineage. Aire has been suggested to be a potential factor implicated in this role. The objective of this study was to characterize Aire and Foxp3 expression in TNC/thymocyte complexes. Methods:Aire and Foxp3 expression was investigated by RT-qPCR in TNC/thymocyte complexes isolated by enzymatic digestion and sedimentation. Aire and Foxp3 proteins were located by confocal microscopy and specific immunocytochemistry. Results: Both Aire and Foxp3 transcripts were detected in TNC/thymocyte complexes. Foxp3 was detected in the nucleus of thymocytes internalized into TNC. Aire was located mainly in TNC cytoplasm and, although to a lower degree, in the nucleus of some TNC-associated thymocytes. Conclusions: Aire and Foxp3 are present in the particular TNC microenvironment which has previously been shown to support thymic selection. The differential localization of these two markers suggests a role for TNC in nTreg development.

Interaction between dendritic cells and nerve fibres in lymphoid organs after oral scrapie exposure

In transmissible spongiform encephalopathies (TSEs), the infectious agent, called PrPsc, an abnormal isoform of the cellular prion protein, accumulates and replicates in lymphoid organs before affecting the nervous system. To clarify the cellular requirements for the neuroinvasion of the scrapie agent from the lymphoid organs to the central nervous system, we have studied, by confocal microscopy, the innervations within Peyer’s patches, mesenteric lymph nodes and the spleen of mice in physiological conditions and after oral exposure to prion. Contacts between nerve fibres and PrPsc-associated cells, dendritic cells (DCs) and follicular dendritic cells (FDCs), were evaluated in preclinical prion-infected mice. Using a double immunolabelling strategy, we demonstrated the lack of innervation of PrPsc-accumulating cells (FDCs). Contacts between nerve fibers and PrPsc-propagating cells (DCs) were detected in T-cell zones and cell-trafficking areas. This supports, for the first time, the possible implication of dendritic cells in the prion neuroinvasion process.

Oral scrapie infection modifies the homeostasis of Peyer’s patches’ dendritic cells

In transmitted prion diseases the immune system supports the replication and the propagation of the pathogenic agent (PrPSc). DCs, which are mobile cells present in large numbers within lymph organs, are suspected to carry prions through the lymphoid system and to transfer them towards the peripheral nervous system. In this study, C57Bl/6 mice were orally inoculated with PrPSc (scrapie strain 139A) and sacrificed at the preclinical stages of the disease. Immunolabelled cryosections of Peyer’s patches were analysed by confocal microscopy. Membrane prion protein expression was studied by flow cytometry. In Peyer’s patches (PP), dissected at day one and day 105 after oral exposure to scrapie, we observed an increased population of DCs localised in the follicular-associated epithelium. On day 105, PrPSc was found in the follicles inside the PP of prion-infected mice. A subset of Peyer’s patches DCs, which did not express cellular prion protein on their surface in non-infected mice conditions, was prion-positive in scrapie conditions. Within Peyer’s patches oral scrapie exposure thus induced modifications of the homeostasis of DCs at the preclinical stages of the disease. These results give new arguments in favour of the implication of DCs in prion diseases.

Adoptive Transfer of T Lymphocytes Sensitized against the Prion Protein Attenuates Prion Invasion in Scrapie-Infected Mice

There is to date no effective way of preventing or curing neurodegenerative diseases such as Alzheimer disease or transmissible spongiform encephalopathies. The idea of treating those conditions by immunological approaches has progressively emerged over the last ten years. Encouraging results have been reported in Alzheimer disease and in peripheral forms of mouse prion diseases following passive injection of Abs or active immunization against the peptides or proteins presumably at the origin of those disorders. Still, major difficulties persist due to some characteristics of those conditions such as slow evolution, brain location, uncertainties regarding precise pathogenic pathways, and, above all, the fact that the target Ag is self, meaning that it is poorly immunogenic and potentially harmful if tolerance was transgressed. To analyze some of those difficulties, we are developing adoptive cell transfer approaches. In this study, lymphocytes sensitized against the prion protein in nontolerant Prnp−/− mice were transferred into histocompatible wild-type recipients which were partly or totally devoid of their own lymphocytes. Under such conditions, we found that the engrafted T lymphocytes resisted peripheral tolerance, remained reactive for several months against epitopes of the prion protein, and significantly attenuated the progression of prions in secondary lymphoid organs with subsequent delay in the evolution of the neurological disease. Interestingly, those protective T lymphocytes secreted lymphokines and migrated more readily into the host CNS but did not appear to be engaged in cooperation with host B cells for Ab production.

9. Germinal centre innervation of bovine and human tonsils related to prion diseases

A number of immunological functions in CD4+ T cells are dependent on the circadian rhythm as we and others could previously demonstrate. Little is known about the underlying mechanisms. One possibility could be the circadian rhythm of the molecular clock in T cells. The molecular clock is known to control the circadian rhythm in the brain and several peripheral organs. To address this question we analyzed the expression of five clock genes (Bmal1, Per2, Cry1, Rev-erba, Dbp), the production of cytokines and the CD40L expression in CD4+ T cells from human volunteers. A total of 15 healthy young men were examined under defined conditions over 24 h in the sleep lab. Venous blood was drawn periodically every 3 h, CD4+ T cells were isolated. T cells were split: one fraction was used for the investigation of clock gene expression and the second fraction was polyclonally stimulated and analyzed applying FACS. We found that on average 32% of polyclonally stimulated highly purified CD4+ T cells express CD40L at 15:00 h whereas only 2% of the CD4+ T cells express CD40L at 6:00 h. Additionally there is also a strong rhythm for the production of INF-g. Furthermore, we have preliminary data demonstrating the rhythmic expression of the core clock genes Bmal1and Cry1 in CD4+ T cells. These findings demonstrate that highly purified CD4+ T cells have a strong functional circadian rhythm and that a possible underlying mechanism could be the molecular clock.