Investigation of the role of the anaphylatoxin receptor C5aR2 in the regulation of NK cell-mediated immune responses

In this project, we aim to investigate the poorly understood role of the complement system in the regulation of NK cells. The complement system is an ancient component of the immune system that was originally thought to play a role only in the clearance of infection. It is now known that the complement system also plays an important role in immune regulation and homeostasis. Recent evidence has shown that the complement system is also important in cancer immunology. However, its role is not fully understood. In this context we now aim to investigate the anaphylatoxin C5a and its receptor C5aR2. In previous work we have shown that this receptor plays an important role in regulating IFN γ expression in NK cells and has an influence on NKp46 expression. The activating receptor NKp46 plays an important role in the control of metastasis by NK cells. Therefore, in this project we plan to investigate this axis in more detail. Preliminary experiments have shown that C5aR2-deficient mice are characterized by NK cells overexpressing NKp46 and that this is protective in an in vivo experimental lung metastasis model of B16-F10 melanoma. In the proposed project, we aim to narrow down this effect to altered NK cell cytotoxic potential in C5aR2-deficient mice, which we will analyze using various in vitro cytotoxicity assays. In these assays we will compare the cytotoxic activity of NK cells from wild-type, systemic and NK cell-specific C5aR2 knock-out, and as a control C5aR1 knock-out mice. In addition, we will use the tdTomato-C5aR2 reporter mouse strain to map the expression of C5aR2 in different immune cell populations during the progression of the experimental B16-F10 lung metastasis model. We will also use lung tissues from this model for spatial transcriptomics to gain detailed insight into differential RNA expression in metastatic and healthy tissues. To determine whether C5aR2 expression in professional immune cells or in non-hematopoietic cells is important for the establishment of experimental lung metastasis, we will generate bone marrow chimeras in which only one of the aforementioned cell populations lacks C5aR2 expression and use these bone marrow chimeras in the in vivo B16-F10 model. In addition, to investigate whether the effects observed in the systemic knockout mice are due to the absence of C5aR2 in the NK cells themselves or in other NK cell influencing cell populations, we will use cell-specific knockouts (e.g. for NK cells or dendritic cells) and subject them to the in vivo B16-F10 model.This project will provide essential new insights into the role of the anaphylatoxin receptor C5aR2 in metastasis formation, in particular highlighting its relevance in the regulation of NK cell functions that mediate the control of metastasis.