Homologous recombination plays a crucial role to repair DNA strand breaks that may occur spontaneously upon replication fork collapse, during the course of radio- or chemotherapy or in a programmed manner during meiosis. Understanding the molecular mechanisms of re-combinational repair is thus very important not only from a basic research viewpoint, but it is also highly relevant for human health. Here, we will define the function of nucleases in homol-ogous recombination. First, we will study the initial steps in this pathway. We could show previously that the S. cerevisiae Sae2 protein promotes the endonuclease activity of the Mre11-Rad50-Xrs2 (MRX) complex near protein blocked DNA ends. This initiates nucleolytic resection of DNA breaks and activates homologous recombination. Our biochemical setup will be instrumental to define how is the activity of Sae2 regulated by phosphorylation on a mech-anistic level and how physiological protein blocks direct the Mre11 endonuclease. We will ex-tend the study to the human system, and attempt to apply the gained knowledge to improve the efficiency of genome editing by activating recombination in conjunction with the CRISPR-Cas9 nuclease system. Second, we will study how homologous recombination promotes gen-eration of genetic diversity during sexual reproduction. DNA strand breaks are introduced in-tentionally during the prophase of the first meiotic division. They are then processed by the recombination machinery into Holliday junction intermediates. These joint molecules are preferentially converted into crossovers in meiosis, resulting in exchange of genetic infor-mation between the maternal and paternal DNA molecules. This is dependent on the Mlh1-Mlh3 nuclease through a yet unknown mechanism. We will study how Mlh1-Mlh3 in complex with other proteins guarantee crossover outcome to promote diversity of the progeny.

In cancer immunotherapy and vaccine field, considerable efforts have been invested to optimize the induction of effector T cells that, by recognizing tumor-specific or pathogen-associated antigens, control tumor cells or infections. Preserving effector T cell function is a major focus of cancer immunotherapy approaches for clinical trials, as is the development of strategies to target regulatory T cells (Tregs) that directly control T cell hypo-responsiveness. In the vaccine field, on the other hand, several strategies have been developed to improve T cell immunogenicity to heterologous antigens expressed by viral vectors. Especially for HIV viral vectors, new vaccine approaches have yielded promising results in primates, although effectiveness was limited in human clinical trials so far. Tumor-associated neutrophils (TAN) participate in the control of human tumor progression. If and how TAN interact with effector Tregs at distinct tumor stages remains to be determined. TAN signals that may regulate the functional state of tumor T cells must be defined. It is also not known whether Tregs interact with TAN and facilitate their functional switch from anti- to pro- tumorigenic state. Distinct neutrophil subtypes are recruited as a result of pro-inflammatory environment during virus infection. Study of the mechanism of neutrophil-dependent control of T cell subset responses to virus-delivered antigens would be of major interest for the generation of viral-based vaccines. The ability of neutrophil subtypes to interact with T cells must be defined to improve the virus-based vaccine efficacy. Our studies could provide: • new treatment strategies that prevent TAN dysfunction, Tregs activation and subsequent effector T cell hyporesponsiveness, and thus increase the effectiveness of cancer immunotherapy • new vaccine approaches to modulate neutrophil subtypes responses to improve antigen-specific T cell responses, and thus increase the effectiveness of HIV vaccines.

Adoptive T cell therapies (ACTs) are emerging as a promising strategy to treat cancer. Tumor-infiltrating lymphocytes (TILs) are expanded ex vivo, selected for recognition of neoantigens, further expanded and then infused back into patients. This procedure requires extensive culturing and expansion of TILs during which many T cell clonotypes are lost. As tumor-reactive TILs are often exhausted and tend to be overgrown by functional, non-specific T cells in culture, the chance to identify potent tumor-reactive T cells dramatically decreases. Moreover, extensive expansion of T cells diminishes their anti-tumor activity and persistence in the body after adoptive transfers. Thus, improving the fitness of T cells is crucial to increase the success rate of ACTs and make this therapy accessible to a broad spectrum of cancer patients. Our first aim is to increase the fitness of T cells by designing metabolic and pharmacological interventions based on proteomic profiles of TILs from patients with liver cancer. Second, we will use machine-learning algorithms for the extraction of signatures to predict whether TILs grow well in culture, require and respond to metabolic interventions, or cannot be revitalized and do not grow at all. To deal with non-growing T cells, we aim at establishing a microfluidics-based workflow to graft the entire T cell receptor (TCR) repertoire from thousands of non-growing TILs onto fast growing Jurkat cells. After selecting Jurkat cells that recognize neoantigens, their TCRs will be expressed on naïve T cells obtained from the patient’s blood that are fit and suitable for ACT. This project will contribute to a better understanding of the T cell response to liver cancer and help increasing the success of personalized ACTs for solid tumors.