The role of Cbl-b for NK cell function

Immune evasion of malignant tumors is the central obstacle for generation of an endogenous and therapeutic anti- tumor immune response. NK cells play a critical role for cancer immune surveillance, and thus are highly attractive immune cells for adoptive cancer therapy. However, in parallel to the limitations in other adoptive immune cell approaches, the cells are in general transferred to the cancer-associated milieu of immune-suppression or unproductive inflammatory response not supporting directed elimination of malignant cells, but rather promoting cancer cell growth. Thus, adoptively transferred cells have to be modified to be resistant to the negative cues exerted by cancer-associated inhibitory factors, such as TGF-ß and regulatory T cells (Treg). It is known from T cells that the E3 ubiquitin ligase Cbl-b acts as a threshold regulator by fine-tuning the activation threshold of T cells. T cells lacking Cbl-b are hyperactive and do not need a co-stimulatory signal for proper activation. We have previously shown that Cbl-b is also highly expressed in NK cells. However, so far it has not been shown to be of functional relevance for NK cell biology. Based on our preliminary data, we are confident that Cbl-b plays a major role in regulating NK cell function. We clearly show that the tumor-rejecting phenotype of cblb-deficient animals can be abolished by depletion of NK1.1 expressing NK cells. Moreover, isolated NK cells from cblb-deficient animals are hyperactive and partially resistant to the suppressive effects induced by TGF-ß when compared to NK cells isolated from wt littermates. Accordingly, knockdown of Cbl-b in a human NK cell line also induces increased killing activity. However, unexpectedly despite the strong functional alterations observed in cblb- deficient NK cells, the relative frequency of CD3+NK1.1+DX5+ NK cells is reduced in cblb-deficient animals, which might point out to a dual and opposing role of Cbl-b in NK cell development versus NK cell function. First data from an in vitro ubiquitination substrate screen identified the TMA receptor family as prime substrate of Cbl- b. Thus, it is the main goal of this project to exactly define the essential and non-redundant role of Cbl-b and its substrates in NK cell biology including various typical biological features of this important immune cell population. A special focus on TGF-ß and Treg sensitivity will answer the question if modulation of Cbl-b might be a rational approach to deprive NK cells from tumor-associated negative cues. This study should set the stage for development and pre-clinical validation of adoptive immune cell transfer protocols approaching the transfer of hyperactive NK or LAK cells for cancer treatment.