Pharmacological treatment of Autoimmune Polyendocrine Syndrome type I with Rapamycin, a mTOR inhibitor

FARM399/05HMATF-FAR

Systematic evaluation of immune regulation and modulation

Cancer immunotherapies are showing promising clinical results in a variety of malignancies. Monitoring the immune as well as the tumor response following these therapies has led to significant advancements in the field. Moreover, the identification and assessment of both predictive and prognostic biomarkers has become a key component to advancing these therapies. Thus, it is critical to develop systematic approaches to monitor the immune response and to interpret the data obtained from these assays. In order to address these issues and make recommendations to the field, the Society for Immunotherapy of Cancer reconvened the Immune Biomarkers Task Force. As a part of this Task Force, Working Group 3 (WG3) consisting of multidisciplinary experts from industry, academia, and government focused on the systematic assessment of immune regulation and modulation. In this review, the tumor microenvironment, microbiome, bone marrow, and adoptively transferred T cells will be used as examples to discuss the type and timing of sample collection. In addition, potential types of measurements, assays, and analyses will be discussed for each sample. Specifically, these recommendations will focus on the unique collection and assay requirements for the analysis of various samples as well as the high-throughput assays to evaluate potential biomarkers

Molecular mechanisms for the regulation of the <i>Il-10</i> gene in CD4 T cells : comparison of IL-10-T_regand CD25+T_reg.

This thesis outlines and provides direct evidence for transcription factor-mediated instructive regulation of the 11-10 gene in CD4 T cells. Firstly, I describe transcription factors that are differentially expressed in populations of IL-10-producing CD4 T cells. Genome-wide mRNA profiles (GeneChip) are used from six populations of CD4 T populations which represent both temporal and lineage-specific time-points within naive, effector and regulatory CD4 T cell populations. Secondly, validation experiments (qPCR) verified whether transcription factors isolated from the genome-wide profiling experiments (GeneChip) were quantitatively expressed in IL-10-producing populations. Thirdly, comparative genomics was used to analyze in silico the 11-10 locus for conserved putative transcription factor binding sites at potential regulatory regions. Selected transcription factors were retro-virally transduced into primary naive CD4 T cells and analyzed for their ability to induce IL-10 expression in a variety of differentiation conditions. Finally, the role of functionally validated transcription factors inducing IL-10 production was investigated at the molecular level, via their presence at specific locations at the 11-10 locus in vivo and in chromatin modifications. One example, GATA-3, shown here to be differentially expressed in IL-10-producing CD4 T cell populations, instructing modifications at the 11-10 locus and inducing IL-10 production in CD4 T cells. The second aim of this thesis was to investigate — at the transcription factor-level -- the comparison of two phenotypically different CD4 regulatory T cell populations (CD25+Treg and IL-10-Treg). On one hand, both CD4 T regulatory cell populations are similar in that they do not produce effector upon secondary stimuli, both are anergic, and both suppress CD4 T cell proliferation in vitro and in vivo (as shown by their ability to abrogate multiple autoimmune and allergic disease animal models). On the other hand, CD25+Treg are phenotypically described by their expression of the lineage-specific transcription factor, FoxP3, and IL-10- Treg do not express FoxP3. Therefore, the molecular mechanisms as to how IL-10- Treg do not express effector cytokines, are anergic and suppress CD4 T cell proliferation in vitro and in vivo without the expression of FoxP3 is investigated in this thesis. This thesis concludes by providing strong evidence that IL-10-Treg down-regulate key NF-kB and AP-1 family transcripts and inhibit NFAT transcriptional activity, thereby compensating for FoxP3 expression, which is necessary in natural occurring CD25+Treg

The influence of secretogranin 2 on the assembly and presentation of the MHC class I complex in melanoma

Melanoma is the reason for the majority of skin cancer-related deaths and reports a rising incidence over the past years. It arises from transformed melanocytes, the melanin-producing skin cells. The primary cause of melanoma is high exposure to ultra violet (UV) radiation. Besides UV radiation, also other factors like genetic predisposition or a weakened immune system can contribute to melanoma development. Depending on the cancer stage, location, and genetic profile different treatment options exist. Gaining new insights into the underlying mechanisms of melanoma progression contributed to the development of new treatment options, such as targeted therapies or immunotherapies. Unfortunately, the development of resistances against treatments negatively influences the therapy success. Therefore, therapy success could be improved through better understanding of the mechanisms leading to therapy resistance and developing potent new therapeutic approaches. Previous work in our laboratory on the histone methyltransferase SETDB1 (SET domain bifurcated histone lysine methyltransferase 1) revealed a possible role of secretogranin II (SCG2) in melanoma pathogenesis. The results showed that high intratumoral SCG2 expression in melanoma patients correlates with poor survival rates. Based on these data, there was reason to further examine the role of SCG2 in melanoma. The aim of this project was to investigate the role of SCG2 in melanoma through identifying mechanisms that are influenced by SCG2. First, I could demonstrate that primary melanoma and melanoma metastases show high SCG2 expression levels. By comparing the gene expression levels between SCG2-overexpressing (OE) and control melanoma cells, I found a downregulation of components of the antigen presenting machinery (APM). The components of the APM are important for the correct assembly of the MHC class I complex. Using flow cytometry analysis, I could show that the surface MHC class I levels were downregulated in SCG2 OE melanoma cells. Due to the downregulation of MHC class I, these cells were less sensitive towards cytotoxic T cell-induced killing. The consequences of SCG2 OE on the expression of several APM components, MHC class I surface presentation, and sensitivity towards cytotoxic T cells could be partially reversed by IFNγ treatment. Taken together, these findings could contribute to the understanding of melanoma immune evasion mechanisms and the role of SCG2 in this process. Consequently, when it comes to the success of checkpoint blockade and adoptive immunotherapy, SCG2 could be a valuable prognostic factor. Also, new insights into the pathways involved in SCG2-induced MHC class I downregulation could open up new possibilities for melanoma treatment

Studies on the molecular and functional properties of exosomes in the metastatic prostate cancer bone microenvironment

Prostate cancer is the most common cancer type and the second leading cancer related cause of death worldwide in men. Prostate cancer patients initially respond to standard treatment (e.g., hormonal, surgery/irradiation) but about 30% of them will develop resistance and progress to metastatic Castration Resistant Prostate Cancer (mCRPC). The cornerstone therapy selection for these patients is chemotherapy (e.g., taxanes). Bone is the most common metastatic site in prostate cancer (mCRPC) and the most frequent cause of death in mCRPC. One of the main bone targeted therapies for patients with bone metastatic CRPC is Radium223, an alpha emitter that has been shown to prolong survival as a single agent. Monitoring the bone tumor microenvironment (bone-TME) is challenging and is based on invasive bone biopsies that cannot be readily performed longitudinally. Liquid biopsies are an attractive approach to monitor the bone-TME including circulating tumor cells, ctDNA and extracellular vesicles (EVs). EVs have a lipid bilayer, contain nucleic acids and proteins. They have been shown to play an important role in homotypic and heterotypic intercellular communication, as well as serve as a source of biomarkers for response and resistance to therapy. In this thesis, the molecular properties of EVs were studied, co-clinically (in vitro, in vivo and in patient samples). In paper I, we characterized the EV transcriptome changes in response to Radium-223 in vivo and patients’ samples. We identified changes in genes related to bone, DNA repair and immune in both the pre-clinical models and patient samples. Treatment with Radium-223 showed a downregulation of bone related transcripts and an upregulation of DNA repair pathways (pharmacodynamic measure). Furthermore, changes in the immune system that are associated with immunosuppression and immune checkpoint activation were identified in patients with unfavorable overall survival. The data obtained indicate that EVs can detect changes in the bone-TME that were functionalized by combining Radium-223 with immunotherapy that improved treatment efficacy. In paper II, we characterized the EV transcriptome for patients treated with Cabazitaxel. Pathway and gene enrichment analysis identified several pathways and associated genes that were enriched in patients that did not respond to Cabazitaxel. Furthermore, at baseline EVs derived from the plasma of Non-responders (NR) were enriched in transcripts encoding genes that are related to oncogenesis, cytoskeleton and immune regulation. Two genes identified to be enriched in NR are Stathmin-1 and ITSN-1 both of which have been previously associated with resistance to Cabazitaxel. Further studies are needed to determine whether longitudinal monitoring of these and other genes identified in the EVs correlates with treatment response and clinical outcome. Taken together, our studies demonstrate that plasma derived EVs could be a useful tool in monitoring the bone TME as well as treatment responses and acquisition of resistance that correlate with clinical outcome