No abstract

The adaptive immune system plays an essential anti-tumor role through immunosurveillance and response to immunotherapies. Characterizing phenotypic features and mechanisms of dysfunction of tumor-specific T cell populations may uncover novel immunotherapeutic targets and biomarkers of response. To study tumor-specific T cell responses in vivo, a tumor model must express a known neoantigen. While transplant models with known neoantigen expression are widely available, autochthonous tumor models in which the tumor coevolves with the immune system are limited. In this study, we combined CRISPR/Cas9 and sleeping beauty transposase technology to develop an autochthonous orthotopic murine sarcoma model with oncogenic KrasG12D, functionally impaired p53, and expression of known MHCI and MHCII sarcoma neoantigens. Using MHC tetramer flow cytometry, we identified a tumor-specific immune response in the peripheral blood as early as 10 days after tumor induction leading to tumor clearance. Tumors developed at high penetrance after co-depletion of CD8 and CD4 T cells, but depletion of either CD8 or CD4 T cells alone was insufficient to permit tumor growth. These results suggest that CD8 and CD4 T cells can independently contribute to immunosurveillance leading to clearance of sarcomas expressing MHCI and MHCII neoantigens.

ATP Synthase is a molecular motor that utilizes a rotary mechanism to synthesize adenosine triphosphate (ATP), the fundamental energy currency of life. The torque for this mechanism is generated in the membrane‐embedded FO motor by harnessing the proton motive force, where protons flow down the electrochemical gradient through two half channels in the motor. The exit half channel on the cytoplasmic side is found at the interface of subunit a (stator) and the subunit c ring (rotor). Previous work on the E. coli ATP synthase has suggested that Thr51 in subunit c is involved in the proton translocation process. To investigate the role of this residue and chemical requirements at this position, we generated six substitution mutants and assayed their in vitro ATP synthesis, H+ pumping, and H+ permeability activities as well as the ability of mutants to carry out oxidative phosphorylation in vivo. Polar and hydrophobic mutations were generally tolerated, except for a notable decrease in function when a negative charge is introduced. Unusually, substitution with Cys completely abolished in vitro functions but only mildly inhibited growth on succinate. Overall, our results suggest that cThr51 may play a supporting role in the interaction between the c‐ring and subunit a in the region of the proton exit channel.

F1Fo ATP synthase is a ubiquitous molecular motor that utilizes a rotary mechanism to synthesize adenosine triphosphate (ATP), the fundamental energy currency of life. The membrane-embedded Fo motor converts the electrochemical gradient of protons into rotation, which is then used to drive the conformational changes in the soluble F1 motor that catalyze ATP synthesis. In E. coli, the Fo motor is composed of a c10 ring (rotor) alongside subunit a (stator), which together provide two aqueous half channels that facilitate proton translocation. Previous work has suggested that Arg50 and Thr51 on the cytoplasmic side of each subunit c are involved in the proton translocation process, and positive charge is conserved in this region of subunit c. To investigate the role of these residues and the chemical requirements for activity at these positions, we generated eleven substitution mutants and assayed their in vitro ATP synthesis, H+ pumping, and passive H+ permeability activities, as well as the ability of mutants to carry out oxidative phosphorylation in vivo. While polar and hydrophobic mutations were generally tolerated in either position, introduction of negative charge caused a substantial defect. We discuss the possible effects of altered electrostatics on the interaction between the rotor and stator, water structure in the aqueous channel, and interaction of the rotor with phospholipids.