Novel immunodeficiency unravels immune homeostasis mechanism

The prevalence of autoimmune and autoinflammatory disorders is continuously rising in the developed world. However, the underlying molecular mechanisms for such altered immune homeostasis resulting in immune response towards self is only barely understood. Genome-wide association studies have uncovered several susceptibility loci for human autoimmune disorders, but such studies often remain associative without mechanistic proof. As an alternative approach, we investigate patients with severe early-onset autoimmune and/or autoinflammatory phenotypes to uncover hitherto unknown monogenetic defects causing such phenotypes, thereby identifying critical mediators of immune homeostasis with important implications on molecular understanding of the critical processes underlying immune homeostasis. As part of these efforts, we recently identified a novel monogenic cause in a syndrome with severe multi-organ autoimmunity, caused by homozygous mutations in a gene encoding a guanine nucleotide exchange factor (GEF) highly expressed in T cells. We could show that the identified mutation leads to a reduction in exchange activity towards the small GTPase CDC42. Furthermore, we could link the mutated GEF and the regulation of cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) expression and trafficking to the outer cell membrane. Both processes are severely reduced in activated T cells of affected individuals and amenable to rescue upon ectopic expression of the wild type allele. Mechanistically, our data suggest an impact on the small GTPase RAB11, which we could identify as a novel interactor of this GEF. In this project, we want to investigate the role of the identified mutated protein in immune homeostasis by defining its role in immune tolerance in molecular detail. Furthermore, we will investigate the impact of the identified mutation on several essential T-cell signaling processes including calcium flux, proliferation, cytokine production and Th1/Th2/Th17 effector cell differentiation. As we could already identify perturbed CTLA-4 expression and trafficking, we further aim to investigate the mechanistic cause for this defect. To this end, we will characterize the RAB11 interaction and investigate the pathway leading to the severe autoimmune phenotype. These biochemical and molecular biology approaches will provide us with the opportunity to validate the relevance of the identified pathway for immune homeostasis. We further will screen a large in-house biobank of genetically undefined PID patients with autoimmunity for defective CTLA-4 trafficking which we will analyze with our well-established genetic pipeline. Collectively, we here identify a hitherto unknown key regulator of immune homoeostasis in humans requiring comprehensive molecular characterization to understand its precise role in the immune system. The proposed research will unravel novel mechanisms of autoimmunity relevant far beyond individual patients, but will substantially contribute to the understanding of immune dysregulation in a significant fraction of autoimmune patients and will potentially open novel targeted therapeutic options not only in the autoimmune field but also in cancer checkpoint blockade.

Orphan diseases of unknown genetic and mechanistic origin, and the consequential lack of personalized treatment options, represent a continuous problem for precision medicine. Inborn errors of the immune system cause defects in immune cell development or in immune control mechanisms, either resulting in life-threatening infections, or in autoimmune reactions due to the inability to distinguish self-tissue from pathogenic (non-self) threats. The study of genetically determined (auto)immune manifestations helps to unravel still poorly understood molecular mechanisms and their consequences for human disease. Due to defined mutations typically affecting a single gene, they are highly beneficial to identify yet unknown molecular players causing (auto)immunity, and to suggest potential therapeutic options. In this FWF-funded work, through next-generation sequencing and molecular cellular investigations, we identified homozygous mutations in the gene DEF6 in patients with systemic autoimmunity as the molecular cause of a novel inborn error of immunity. We found that due to mutated DEF6 protein, the availability of the checkpoint receptor CTLA-4 on T-cell surfaces, a key inhibitory control factor of T-cell stimulation, is compromised. As a consequence, due to mutated DEF6, CTLA-4 cannot dampen immune responses after prolonged stimulation, and this defect results in autoimmune reactions. In detail, we identified a novel molecular mechanism by which DEF6 ensures CTLA-4 availability. CTLA-4 itself has been implicated in disease, requiring tightly balanced presence on T cells for its inhibitory action. Too little CTLA-4 expression causes autoimmunity, while its pharmacological blockade is highly beneficial in cancer immunotherapy. Yet the cellular components regulating CTLA-4 remain poorly understood. With DEF6, we here identified a novel determinant of CTLA-4 homeostasis, allowing for a better understanding of autoimmune pathogenicity with direct implications in therapy. Resulting from our study, CTLA-4-Ig administration has been initiated in one of the DEF6-deficient patients, achieving sustained remission of symptoms.