Analysis of the TGFß-receptor/Cbl-b pathway in autoimmunity and tumor immunity

T lymphocytes represent an important branch of the adaptive immune system that fights pathogens like bacteria and viruses and can also attack tumor cells to retard tumor growth or even reject established tumors. On the other hand, inappropriate immune reactions can lead to autoimmune diseases like multiple sclerosis or lupus erythematosus. Therefore, it is crucial that there is an intrinsic fine tuning to activate the immune system only when it is needed but to silence it when it is potentially harmful. Cytokines of the transforming growth factor ß (TGFß) family play important roles in many cellular functions. TGFß1 is predominantly expressed in the immune system and is one of the key regulators of immune homeostasis and peripheral T cell tolerance. TGFß1 has been shown to suppress T cell activity, thereby limiting autoimmunity. On the other hand, TGFß1 has been found to be secreted into the tumor microenvironment of many malignancies and to contribute essentially to immune evasion of tumors. In addition to the immunosuppressive properties of TGFß1, this cytokine is pivotal for T cell differentiation into diverse effector T cell subsets. Cbl-b is an E3 ubiquitin ligase that regulates T cell activation thresholds by mediating the requirement for CD28 costimulation. Furthermore, Cbl-b has also been shown to mediate the suppressive effects of TGFß1. Cblb-deficient T cells are less sensitive to TGFß1 and to inhibition by regulatory T cells. Moreover, cblb-/- mice display enhanced responses to a TGFß1-secreting tumor compared to WT mice. However, despite the striking phenotype of cblb -/- mice, little is known about the physiological, disease-preventing role of Cbl-b in the TGFß receptor pathway in effector T cells so far. Here we aim to better understand the signaling cascades leading to immune tolerance or on the other hand autoimmunityumor immunity, focusing on the role of the TGFßR/Cbl-b/Smad pathway, which was recently revealed by our laboratory. Our project will address the following main topics: 1.) Regulation of the Cbl-b-dependent ubiquitination and degradation of Smad7. Nuclear role of Cbl-b. Evaluation of additional Cbl-b functions in the TGFßRsignaling pathway (besides its proposed role in Smad signaling). Specifically: Regulation of GM-CSF production by Cbl-b. 2.) Role of the Cbl-b/Smad axis in the development of autoimmune diseases as a consequence of altered TGFß sensitivity of T cells. 3.) Role of the Cbl-b/Smad axis in the prevention of effective anti-tumor responses as a consequence of an immunosuppressive tumor milieu generated by TGFß. The understanding of physiological Cbl-b functions in the TGFß receptor pathway would represent a major step in T cell research. Exploring the mechanisms which enforce immune tolerance is of interest because it may open the possibility to manipulate T cell functions in disease or tumor immunity.

T cells represent an important branch of the adaptive immune system that fights pathogens like bacteria and viruses and can also attack tumor cells to retard tumor growth or even reject established tumors. On the other hand, inappropriate immune reactions can lead to autoimmune diseases like multiple sclerosis. Therefore, it is crucial that there is an intrinsic fine tuning to activate the immune system only when it is needed but to silence it when it is potentially harmful. The research group led by Ass.-Prof. Dr. Thomas Gruber at the Division of Cell Genetics (Medical University Innsbruck) has a focus on the molecular "brakes" that dampen T cell activity in order to prevent autoimmunity. However, these "brakes" (also termed "checkpoints") hamper efficient anti-tumor T cell reactions as well. Cbl-b, a protein of the family of E3 Ubiquitin ligases that is expressed in T cells, has been previously shown to inhibit activation of these cells potently. Genetically modified mice deficient in Cbl-b are more susceptible to autoimmune disorders but also reject tumors efficiently. In the course of the project funded by the FWF, the group of Dr. Gruber could now reveal two molecular mechanisms by which Cbl-b inhibits autoimmunity and attenuates anti-tumor immunity. In the tumor immunity part of the project, it could be shown that Cbl-b mediates the inhibitory function of the PD-1 receptor in T cells. In corresponding experiments, T cells without Cbl-b were resistant to inhibition by the PD-L1/PD-1 interaction. This finding has implications for tumor immunology, as PD-1 is an important immuno-checkpoint whose blockade leads to increased rejection of tumor cells by T cells. This strategy has been in the clinic for several years with the antibody nivolumab. Our in vivo experiments revealed that inhibition of PD-1 in Cbl-b deficient mice has no effect in contrast to wild-type animals. This opens up the opportunity to develop Cbl-b as a biomarker for cancer patients receiving anti-PD-1 therapy. It would then have to benefit first and foremost those patients whose Cbl-b expression is high. This approach (Cbl-b as biomarker) is currently being pursued at the Division of Cell Genetics. In the autoimmunity part of the project, the group around Dr. Gruber demonstrated that Cbl-b regulates the cytokines GM-CSF and Il-3 via the transcription factor NF-B. GM-CSF plays a particularly important role in multiple sclerosis. In a mouse model of this autoimmune disease, the symptoms were significantly worsened in animals without Cbl-b compared to wild-type animals. This effect could be completely reversed by neutralizing GM-CSF with an antibody. This finding may lead to Cbl-b being used as a biomarker in multiple sclerosis therapy. In this case, patients with low levels of Cbl-b would be particularly suitable for anti- GM-CSF therapy. Further research in the laboratory of Dr. Gruber will show if this approach is promising.