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Foxp3+ regulatory T cells are well-known immune suppressor cells in various settings. In this study, we provide evidence that knockout of the relB gene in dendritic cells (DCs) of C57BL/6 mice results in a spontaneous and systemic accumulation of Foxp3+ T regulatory T cells (Tregs) partially at the expense of microbiota-reactive Tregs. Deletion of nfkb2 does not fully recapitulate this phenotype, indicating that alternative NF-κB activation via the RelB/p52 complex is not solely responsible for Treg accumulation. Deletion of RelB in DCs further results in an impaired oral tolerance induction and a marked type 2 immune bias among accumulated Foxp3+ Tregs reminiscent of a tissue Treg signature. Tissue Tregs were fully functional, expanded independently of IL-33, and led to an almost complete Treg-dependent protection from experimental autoimmune encephalomyelitis. Thus, we provide clear evidence that RelB-dependent pathways regulate the capacity of DCs to quantitatively and qualitatively impact on Treg biology and constitute an attractive target for treatment of autoimmune diseases but may come at risk for reduced immune tolerance in the intestinal tract.

CYLD is a deubiquitinating enzyme and acts as a negative regulator of NF-κB signaling. To analyse the function of CYLD in vivo we used CYLDex7/8 mice, which are characterized by loss of the full-length transcript and overexpression of a short splice variant of cyld (sCYLD). In these mice thymic and peripheral CD4 and CD8 T cells were reduced compared to controls. Hence, we were interested to investigate the contribution of CYLD in positive and negative selection in the thymus in vivo. For this purpose we crossed the HY-TCR transgene (HYtg) to CYLDex7/8 mice. The analysis of CYLDex7/8 HYtg males revealed an increase in CD4+CD8+ DP as well as in CD8 SP thymocytes, suggesting a less pronounced negative selection in CYLD mutant mice compared to HYtg control mice. Since medullary thymic epithelial cells (mTECs) play an essential role in the negative selection process of thymocytes, the levels of mTECs in the medullary compartment were examined by histology and FACS analysis. We observed reduced numbers of mTECs, combined with decreased expression levels of the mTEC markers UEA-1, keratin-5, claudin-3 and claudin-4. Interestingly, specifically immature mTECs were significantly reduced compared to controls, suggesting a developmental block in the transition of immature to mature mTECs. Taken together, these results display a crucial role of sCYLD in the development and maturation of mTECs.

CYLD is a tumour suppressor gene which plays an important role in NF-kB signaling. To analyze the function of CYLD in vivo we used the CYLDex7/8 mouse strain, which is characterized by loss of the full-length transcript and overexpression of a naturally occurring short splice variant of the cyld gene (sCYLD). This overexpression resulted in splenomegaly and lymphadenopathy. Further, the B cell numbers in spleen and lymph nodes were increased at the expense of T cells. CYLDex7/8 mice showed a strong reduction of CD4 single positive (SP) and CD8 SP T cells in the thymus and periphery. In in vitro and in vivo studies we could show that CD4+ T cells display a hyperactive phenotype accompanied with an increased production of inflammatory cytokines. We could also demonstrate a defect in negative selection of CYLDex7/8 thymocytes by using the HY-TCR transgenic system. mTECs in the thymus, which are important for the negative selection of autoreactive T cells, were drastically reduced in number. Since abnormal T cell responses are often associated with chronic inflammations, we examined if CYLDex7/8 mice develop immunological abnormalities. Importantly, we could observe that CYLDex7/8 mice crossed onto TCR transgenic background developed severe colonic inflammation accompanied with high production of nuclear autoantibodies.