LAUSR

Background Overexpression of TAM receptors, including MERTK, in some cancers are integral for chemoresistance, proliferation and metastasis.1 Our group has previously demonstrated that T cells also express MERTK and engagement of MERTK signaling is responsible for increased proliferation, functional capacity and metabolic fitness.2 It is therefore important to further study the effect of MERTK inhibition on T cell function in the context of cancer treatments where MERTK inhibitors may play a role. Here we provide evidence that MERTK inhibition impacts greatly on T cell proliferation, specifically reducing phosphorylated mTOR. We have also demonstrated that MERTK expression is increased on CD8 central memory subsets during longterm expansion providing evidence that this signaling pathway may be important for sustaining T memory responses. Materials and Methods Flow cytometric analysis was used to investigate the effect of titration of MERTK small molecule inhibitor UNC2025 on healthy donor T cells activated with CD3/CD28 dynabeads. Cell trace dye was used to track proliferation of CD4 and CD8 T cells along with markers of memory differentiation (CCR7 and CD45RO), activation (CD137) and function (IFNy, Tnfa and IL-2). MERTK signaling was assessed using phospho flow cytometric methodology of phosphorylated mTOR, AKT, ERK1/2, p38-MAPK and STAT5. Long term cultures of donor T cells of up to 28 days were investigated for MERTK expression alongside memory differentiation. Results We demonstrated that moderate concentrations of MERTK inhibitor reduced proliferation of activated T cells. Despite inhibition of cell division, cell size still increased 2 fold compared to resting cells and cell viability remained unchanged. Additionally, the proportion of central memory to effector memory populations and intracellular cytokine production was not impacted. Analysis of molecules involved in MERTK signaling revealed that phosphorylated mTOR was significantly modulated following the addition of MERTK inhibitor. Long term culture of CD8 T cells demonstrated MERTK was significantly increased following early and late re-stimulation, and expression of MERTK was strongly associated with central memory subsets. Conclusions Our results demonstrate that inhibition of MERTK signaling on T cells reduces cell division where mTOR is significantly impacted. Despite this, other functional aspects, such as intracellular cytokine production remain unchanged. Therefore, interruption of MERTK signaling on T cells has a specific effect on cell division rather than cytotoxic function on a cell by cell basis. This has potential ramifications on the use of MERTK inhibitors to treat tumors where the ability to form substantial cytotoxic T cell populations might be reduced. In addition, increased MERTK expression on central memory subsets during long term culture suggests this signaling pathway could be critical for generating memory pools of T cells and provide new avenues for the improvement of adoptive T cell therapy protocols. References Cummings CT, Deryckere D, Earp HS, Graham DK. Molecular pathways: MERTK signaling in cancer. Clin Cancer Res 2013;19(19):5275–5280. Peeters MJW, Dulkeviciute D, Draghi A, et al. MERTK Acts as a Costimulatory Receptor on Human CD8+T Cells. Cancer Immunol Res 2019;7(9):1472–1484. Disclosure Information R.M. Powell: None. M.J.W. Peeters: None. A. Rachbech: None. P.T. Straten: None.