Orphan receptor NR2F6 as a negative regulator of T cell effector functions

Our group recently identified the role of NR2F6 in the transcriptional regulation of immune response thresholds, particularly in the Th17 lineage, critical in autoimmune pathologies 1. Mechanistically, NR2F6 potently antagonizes the ability of nave and Th17 CD4+ T cells to induce expression of the signature cytokines Il2 and Il17. In Th17 cells differentiated and activated ex vivo, loss of Nr2f6 results in amplified NFAT DNA binding at the Il17a promoter and subsequently increased IL-17 transcription. Consistently, Nr2f6-deficient mice have hyper- reactive lymphocytes, develop late-onset immune-pathologies and are hyper-susceptible to the Th17-dependent model of experimental autoimmune encephalomyelitis (EAE). Our previous work has established NR2F6 as PKC substrate and critical transcriptional repressor of adaptive immune responses however the precise mode of regulation and action of NR2F6 as T cell intrinsic negative regulator of effector responses remain unresolved. This project specializes in the composite analysis of NR2F6-selective signal transduction in antigen stimulated T lymphocytes, with a special focus on Th2 and Th17 CD4+ T cell subsets and their key signature cytokine promoters as Il2, Il17a, Il21, and Il4. I propose to address following key questions: Which signalling proteins directly interact with NR2F6 upon antigen receptor stimulation? What are the essential co-regulators (co- repressors/co-activators) and how do these NR2F6:protein interactions contribute to transcriptional threshold regulation on those critical cytokine gene promoters? As Nr2f6 is most abundant expressed not only in the Th17 but also in the Th2 subset, I propose to investigate the potential role of Nr2f6 in the Th2 subset in parallel. In the long run, I would like to start a rational based search to identify additional NR2F6 regulated promoter regions within the Th17 and Th2 subset employing the OTII TCR transgenic mouse model as well as primary human T cells. The use of the two complementary genetic mouse models (Nr2f6 knockout versus NR2F6 over-expressing transgenic mice), NR2F6 siRNA-mediated knockdown approaches in combination with tools in biochemistry and cell biology (visualization of sub-cellular location kinetics) should aid to the elucidation of NR2F6 function in T cells. Dissecting the upstream and downstream signalling mechanisms of NR2F6 in antigen receptor signalling in Th17 as well the Th2 cells may contribute to an improved understanding of the molecular and cellular processes that lead to inflammation and autoimmune disease.

Immune responses protect us against colonization of foreign organisms as bacteria, protozoa, virus or fungi as well as against potential tumor cells. In order to face these challenges immune cells are equipped with highly dangerous weapons which can destroy and even kill own tissue. Therefore it is highly relevant to understand the molecular mechanisms of both activating as well as inhibitory signaling cascades especially in the T cell compartment of the adaptive immune system. Autoimmune diseases as multiple sclerosis, psoriasis, rheumatoid arthritis or diabetes are characterized by the failure of an organism in recognizing its own constituent parts as self, which triggers an immune response against its own tissues. This immune response is often dependent on the T lymphocyte helper subset Th17. Family members of the steroid/thyroid hormone family as e.g. the glucocorticoid receptor are well known for sensing external cues (such as hormones) in order to regulate the expression of specific genes thereby controlling immune reaction of the organism. We could identify a completely new role within the immune compartment for the nuclear orphan receptor NR2F6 and unravel the molecular mechanisms how NR2F6 attenuates T cell receptor thresholds in nave as well in Th17 CD4+ lymphocytes. NR2F6 inhibits transcription of cytokines via occupying DNA binding sites necessary for activating transcription. Only after optimal TCR stimulation NR2F6 is phosphorylated in the nucleus and gives way for the binding of activating transcription factors. In the autoimmune prone subset of CD4+ Th17 cells NR2F6 interferes with the DNA accessibility of NFAT and RORgamma-t the lineage specific transcription factor of CD4+ Th17 cells, ensuring the proper level of Il17a promoter activation. Given the reported key role of the cytokine IL-17 in autoimmune diseases, our report closes an important gap in the understanding of how the expression of this cytokine is controlled via NR2F6. These findings support the idea that selective inhibition of NR2F6 phosphorylation could represent an innovative therapeutic regimen for attenuating IL-17A production as a treatment for certain Th17-mediated autoimmune disorders.