We are interested in understanding the multi-layered tolerance system that prohibits autoimmunity and allergy while retaining the ability to mount an effective immune response against pathogens and cancer. We study how thymic tolerance is achieved and how tolerance is broken in the periphery (Sledzinska et al. 2013). We also investigate how breaking tolerance can be used to treat cancer. Central tolerance in the thymus prevents the maturationof autoreactive T cells. While the general concept of negative selection is clear, it is not understood what gives autoreactive thymocytes the ability to commit suicide. We have generated a mouse model in which we can induce the expression of the T cell receptor and follow a cohort of cells through development. This approach allows direct comparison of cells in different selecting and non-selecting environments. In another approach we use the CRISPR/Cas9 technology to screen for and identify genes relevant for thymic negative selection (Beil-Wagner et al. submitted). The same technology is applied to confirm the in vitro results in vivo by rapid generation of gene- modified animals in our facility. To study the underlying causes of tolerance breakdown we use the mouse models of multiple sclerosis and allergic asthma. We want to understand why in some individuals these diseases develop (and why not in others). Our focus lies on the critical first steps of the developing diseases: antigenic and innate immune stimulation and immune suppressive checkpoints. We have been studying in detail the role of antigen release from dying oligodendrocytes (Locatelli et al. 2012). Currently we investigate the role of pattern recognition receptors such as the TLR system and specific alarmins by breeding together multiple deficiencies or even create them through CRISPR/Cas9 when the genes are clustered on one chromosome. While in autoimmunity and allergy the breakdown of tolerance constitutes a pathological process, such a breakdown can be hijacked for the use in cancer therapy (cancer vaccination, checkpoint inhibitors).