Identifying genetic determinants of T cell-dependent autoimmunity using forward genetics

Rheumatoid arthritis (RA) affects 0.5-1% of the population and is an important health and socioeconomic problem. RA has a high degree of heritability. Thus, extensive efforts have been made to better understand the genetic variability contributing to disease susceptibility. However, dissecting the genetic component of RA in humans has been difficult due to heterogeneity in the human population, multiple testing issues, and lack of accessibility to relevant tissues for proof-of-concept studies. Genetic studies in mouse model systems circumvent these problems, enhancing the possibility to identify disease regulating genetic variants. Here, we use a forward genetics approach in mice to identify and characterize genetic determinants of RA and related autoimmune diseases. First, we have mapped quantitative trait loci (QTL) regulating experimental arthritis using linkage analysis, and then isolated these QTL in congenic strains for in depth functional characterization. Using this approach, we make several important observations. In the first study, we find that promoter polymorphisms regulating expression of the vitamin D receptor affect T cell activation and T cell-driven collagen-induced arthritis. These findings are particularly interesting considering the long-standing association between serum vitamin D and several autoimmune diseases. In the second study, we discover a spontaneous insertion of a long terminal repeat which leads to a deficiency in the SH3GL1 gene (Endophilin A2), protecting mice in several arthritis models. We are first to identify the immunomodulatory properties of SH3GL1, which may prove to be a valuable therapeutic target. In the third study, we identify a polymorphic estrogen receptor binding site that regulates susceptibility to experimental arthritis and other autoimmune models by interfering with estrogen regulation of the T cell marker CD2. These results suggest an important role for CD2 and estrogen in shaping the sexually dimorphic immune response. Collectively, our findings make a significant contribution towards the understanding of RA genetics while demonstrating the value of animal models

Vitamin D3 receptor polymorphisms regulate T cells and T cell-dependent inflammatory diseases

It has proven difficult to identify the underlying genes in complex autoimmune diseases. Here, we use forward genetics to identify polymorphisms in the vitamin D receptor gene (Vdr) promoter, controlling Vdr expression and T cell activation. We isolated these polymorphisms in a congenic mouse line, allowing us to study the immunomodulatory properties of VDR in a physiological context. Congenic mice overexpressed VDR selectively in T cells, and thus did not suffer from calcemic effects. VDR overexpression resulted in an enhanced antigen-specific T cell response and more severe autoimmune phenotypes. In contrast, vitamin D3-deficiency inhibited T cell responses and protected mice from developing autoimmune arthritis. Our observations are likely translatable to humans, as Vdr is overexpressed in rheumatic joints. Genetic control of VDR availability codetermines the proinflammatory behavior of T cells, suggesting that increased presence of VDR at the site of inflammation might limit the antiinflammatory properties of its ligand

Polymorphic estrogen receptor binding site causes Cd2-dependent sex bias in the susceptibility to autoimmune diseases

Complex autoimmune diseases are sexually dimorphic. An interplay between predisposing genetics and sex-related factors probably controls the sex discrepancy in the immune response, but the underlying mechanisms are unclear. Here we positionally identify a polymorphic estrogen receptor binding site that regulates Cd2 expression, leading to female-specific differences in T cell-dependent mouse models of autoimmunity. Female mice with reduced Cd2 expression have impaired autoreactive T cell responses. T cells lacking Cd2 costimulation upregulate inhibitory Lag-3. These findings help explain sexual dimorphism in human autoimmunity, as we find that CD2 polymorphisms are associated with rheumatoid arthritis and 17-β-estradiol-regulation of CD2 is conserved in human T cells. Hormonal regulation of CD2 might have implications for CD2-targeted therapy, as anti-Cd2 treatment more potently affects T cells in female mice. These results demonstrate the relevance of sex-genotype interactions, providing strong evidence for CD2 as a sex-sensitive predisposing factor in autoimmunity

Glycan Activation of Clec4b Induces Reactive Oxygen Species Protecting against Neutrophilia and Arthritis

Animal models for complex diseases are needed to position and analyze the function of interacting genes. Previous positional cloning identified Ncf1 and Clec4b to be major regulators of arthritis models in rats. Here, we investigate epistasis between Ncf1 and Clec4b, two major regulators of arthritis in rats. We find that Clec4b and Ncf1 exert an additive effect on arthritis given by their joint ability to regulate neutrophils. Both genes are highly expressed in neutrophils, together regulating neutrophil availability and their capacity to generate reactive oxygen species. Using a glycan array, we identify key ligands of Clec4b and demonstrate that Clec4b-specific stimulation triggers neutrophils into oxidative burst. Our observations highlight Clec4b as an important regulator of neutrophils and demonstrate how epistatic interactions affect the susceptibility to, and severity of, autoimmune arthritis

Endophilin A2 deficiency protects rodents from autoimmune arthritis by modulating T cell activation

The introduction of the CTLA-4 recombinant fusion protein has demonstrated therapeutic effects by selectively modulating T-cell activation in rheumatoid arthritis. Here we show, using a forward genetic approach, that a mutation in the SH3gl1 gene encoding the endocytic protein Endophilin A2 is associated with the development of arthritis in rodents. Defective expression of SH3gl1 affects T cell effector functions and alters the activation threshold of autoreactive T cells, thereby leading to complete protection from chronic autoimmune inflammatory disease in both mice and rats. We further show that SH3GL1 regulates human T cell signaling and T cell receptor internalization, and its expression is upregulated in rheumatoid arthritis patients. Collectively our data identify SH3GL1 as a key regulator of T cell activation, and as a potential target for treatment of autoimmune diseases

A subset of antibodies targeting citrullinated proteins confers protection from rheumatoid arthritis

Although anti-citrullinated protein antibodies (ACPAs) are a hallmark of rheumatoid arthritis and generally considered pathogenic, their functional relevance is incompletely understood. In this study, the authors describe an ACPA with a protective effect against antibody-induced arthritis in mice

Sanmarco et al., 2023.pdf

Dendritic cells (DCs) control the generation of self-reactive pathogenic T cells. Thus, DCs are considered attractive therapeutic targets for autoimmune diseases. Using single-cell and bulk transcriptional and metabolic analyses in combination with cell-specific gene perturbation studies we identified a negative feedback regulatory pathway that operates in DCs to limit immunopathology. Specifically, we found that lactate, produced by activated DCs and other immune cells, boosts NDUFA4L2 expression through a mechanism mediated by HIF-1a. NDUFA4L2 limits the production of mitochondrial reactive oxygen species that activate XBP1-driven transcriptional modules in DCs involved in the control of pathogenic autoimmune T cells. Moreover, we engineered a probiotic that produces lactate and suppresses T-cell autoimmunity in the central nervous system via the activation of HIF-1a/NDUFA4L2 signaling in DCs. In summary, we identified an immunometabolic pathway that regulates DC function, and developed a synthetic probiotic for its therapeutic activation. </p