Periodic Reporting for period 2 - DANTE (Use of novel techniques to identify pathogenic versus non-pathogenic TH17 cells in experimental autoimmune encephalomyelitis, a murine model of multiple sclerosis.)
Reporting period: 2018-10-01 to 2019-09-30
Autoimmune diseases and cancer are crucial medical conditions of our time. Despite various fundamental therapeutic advances in recent years there is unparalleled wealth to be gained from enhancing our scientific knowledge about pathomechanisms that open doors to precision medicine and targeted therapy. Among the major breakthroughs of the last decade, which made it from the basic research laboratory bench to the patient’s bedside, are: 1) the identification of a new pro-inflammatory T helper subpopulation called Th17 cells which are linked to the pathogenesis of many autoimmune diseases and 2) the identification of regulators of the immune system, so called checkpoint molecules, which determine the functional state of T cells that are critical for the protection against cancer. However, there are case reports that therapies targeting Th17 cells in autoimmune diseases have pro-tumor effects and that therapies used to block or stimulate immune checkpoints to target cancer cells can induce autoimmune disease entities. In this project we therefore wanted to further elucidate how Th17 cells and immune checkpoints are entangled. First, we were able to uncover a completely new function of the well-known cell death receptor Fas. Independently of its function in the induction of cell death, we could show that Fas promotes the differentiation of T cells into Th17 cells by preventing the activation of an important transcription factor of a different T helper subset, Th1 cells. This finding improves our understanding of the mechanism by which Th17 cells become pathogenic. Secondly, using the cytokine Interleukin-27, which can exhibit anti-inflammatory properties and promote tumor growth, we identified two new potential immune checkpoint receptors and studied their function in the context of cancer and autoimmune diseases. GPR65 is a receptor than can sense an acid extracellular environment, which is common in areas of inflammation and in the tumor microenvironment. Without GPR65 being expressed we saw that experimental tumor growth was significantly diminished. Additionally, we detected that Th17 cells deficient of GPR65 produce significantly less pro-inflammatory, potentially autoimmune disease causing cytokines. CysLTR2 is a receptor that binds leukotrienes, known inflammatory mediators, previously linked to allergic diseases like bronchial asthma. We were able to demonstrate that deficiency of CysLTR2 protects from experimental autoimmune encephalomyelitis, a disease model resembling human multiple sclerosis. Antibodies against GPR65 and CysLTR2 could therefore, respectively used in the right setting, serve as promising targets to treat cancer or autoimmune diseases in the future.
Interleukin-17 (IL-17) producing CD4+ T helper cells (Th17) cells are linked to the pathogenesis of highly prevalent inflammatory diseases like multiple sclerosis (MS) and rheumatoid arthritis (RA). To further support the role of IL-17 and Th17 cells in MS, a recent small clinical trial with anti-IL-17 (AIN457, Secukinumab) conducted by Novartis, has shown beneficial clinical effects in reducing the lesion load and clinical disease in MS patients (5, 6). In addition to MS, Th17 cells were also found to be associated with other autoimmune and chronic inflammatory diseases such as RA, ankylosing spondylitis (AS), inflammatory bowl disease (IBD), and psoriasis (7, 8). Therefore, IL-17 and Th17 cells are not only critical for studies of MS but also in other human autoimmune diseases, as in vivo neutralization of IL-17 has also shown high degree of clinical efficacy in both psoriasis and AS (9, 10). Although IL-17 and IL-23-Receptor expressing Th17 cells have been genetically linked to IBD, administration of the same anti-IL-17 antibody (AIN457, Secukinumab) in IBD patients did not inhibit the disease but if anything appeared to worsen the disease (11). This raises the issue of whether all Th17 cells are pathogenic and thus “en-block” deletion of Th17 cells or complete neutralization of IL-17 may result in the loss of tissue-protective Th17 cells. Indeed, it is now appreciated that not all Th17 cells are pathogenic. Recent discoveries imply that two different subtypes of Th17 cells, one that induces autoimmunity (pathogenic) and one that does not (non-pathogenic), cause this observed dichotomy. Possible findings that allow specific targeting of pathogenic Th17 cells would have major translational and therapeutic implications.
To date, the expression and function of co-inhibitory receptors has been most extensively described in CD8+ T cells, where the accumulation and overexpression of these co-inhibitory receptors is known to promote T cell dysfunction and exhaustion. This exhausted cell state is usually characterized by diverse deficits in effector function and leads to an impaired ability to clear chronic viral infections and cancer (14). In contrast, the role of co-inhibitory receptors in CD4+ T cell immunology is not as comprehensively studied however, classic co-inhibitory receptors, such as such as CTLA4 and PD-1, and their respective ligands have already been implicated in regulating differentiation and function of CD4+ T helper subsets, including Th17 cells (15, 16). Data show that e.g. the PD-1 pathway considerably influences Th17 cells by promoting their differentiation, but limiting their response during the effector phase. This is in line with increasing reports of manifestations of autoimmune like diseases in patients who receive cancer immunotherapies that block co-inhibitory receptors like CTLA-4 and PD-1 (17). While this therapy is usually aimed to restore the impaired CD8+ T cell function and restore anti-tumor immunity, the checkpoint blockade - directly or indirectly - appears to initiate the activity of pro-inflammatory CD4+ T cells.
This research project offered the unique opportunity to explore completely newly identified co-inhibitory receptors. To discover their role in regulating Th17 pathology and tumor immunity, we chose two promising G-coupled receptors that could potentially serve as direct targets for medical therapy: GPR65 and CysLTR2. We aimed to analyze whether they not only inhibit pathogenic Th17 cells but also promote differentiation of non-pathogenic Th17 cells. Further, we wanted to examine whether changes to the pathogenic signature of Th17 cells induced by co-inhibitory receptors also translated into altered pathogenicity in vivo. It was also of high interest to us to test how the expression of these newly discovered co-inhibitory receptors synergizes with other co-inhibitory receptors and how they can influence tumor immunity.
To date, the expression and function of co-inhibitory receptors has been most extensively described in CD8+ T cells, where the accumulation and overexpression of these co-inhibitory receptors is known to promote T cell dysfunction and exhaustion. This exhausted cell state is usually characterized by diverse deficits in effector function and leads to an impaired ability to clear chronic viral infections and cancer (14). In contrast, the role of co-inhibitory receptors in CD4+ T cell immunology is not as comprehensively studied however, classic co-inhibitory receptors, such as such as CTLA4 and PD-1, and their respective ligands have already been implicated in regulating differentiation and function of CD4+ T helper subsets, including Th17 cells (15, 16). Data show that e.g. the PD-1 pathway considerably influences Th17 cells by promoting their differentiation, but limiting their response during the effector phase. This is in line with increasing reports of manifestations of autoimmune like diseases in patients who receive cancer immunotherapies that block co-inhibitory receptors like CTLA-4 and PD-1 (17). While this therapy is usually aimed to restore the impaired CD8+ T cell function and restore anti-tumor immunity, the checkpoint blockade - directly or indirectly - appears to initiate the activity of pro-inflammatory CD4+ T cells.
This research project offered the unique opportunity to explore completely newly identified co-inhibitory receptors. To discover their role in regulating Th17 pathology and tumor immunity, we chose two promising G-coupled receptors that could potentially serve as direct targets for medical therapy: GPR65 and CysLTR2. We aimed to analyze whether they not only inhibit pathogenic Th17 cells but also promote differentiation of non-pathogenic Th17 cells. Further, we wanted to examine whether changes to the pathogenic signature of Th17 cells induced by co-inhibitory receptors also translated into altered pathogenicity in vivo. It was also of high interest to us to test how the expression of these newly discovered co-inhibitory receptors synergizes with other co-inhibitory receptors and how they can influence tumor immunity.
Throughout this project I utilized NanoString, popRNAseq, single cell RNA seq analysis to identify a gene signature that differentiates pathogenic from non-pathogenic TH17 cells. The studies carried out during this fellowship demonstrated that not all differentially expressed molecules on pathogenic Th17 cells contribute to pathogenicity and opens the path to generate biologics for the treatment of cancer that does not lead to immune related adverse events (irAEs). Single cell RNA-seq, a technique in which I was trained, has provided new granularity in identifying novel regulators that do not impact TH17 differentiation but regulate the functional state (pathogenic vs. non-pathogenic) of TH17 cells. This technology is one I will utilize in my future institute in Europe.