Peptides presented by MHC class I molecules are mostly derived from proteins synthesized by the antigen‐presenting cell itself, while peptides presented by MHC class II molecules are predominantly from materials acquired by endocytosis. External antigens can also be presented by MHC class I molecules in a process referred to as cross‐presentation. Here, we report that mouse dendritic cell (DC) engagement to a phagocytic target alters endocytic processing and inhibits the proteolytic activities. During phagocytosis, endosome maturation is delayed, shows less progression toward the lysosome, and the endocytosed soluble antigen is targeted for MHC class I cross‐presentation. The antigen processing in these arrested endosomes is under the control of NAPDH oxidase associated ROS. We also show that cathepsin S is responsible for the generation of the MHC class I epitope. Taken together, our results suggest that in addition to solid structure uptake, DC phagocytosis simultaneously modifies the kinetics of endosomal trafficking and maturation. As a consequence, external soluble antigens are targeted into the MHC class I cross‐presentation pathway.
Abstract Transgenic mouse models are widely used in biomedical research; however, current techniques for producing transgenic mice are limited due to the unpredictable nature of transgene expression. Here, we report a novel, highly efficient technique for the generation of transgenic mice with single-copy integration of the transgene and guaranteed expression of the gene-of-interest (GOI). We refer to this technique as f unctionally e nriched ES cell t ransgenics, or FEEST. ES cells harboring an inducible Cre gene enabled the efficient selection of transgenic ES cell clones using hygromycin before Cre-mediated recombination. Expression of the GOI was confirmed by assaying for the GFP after Cre recombination. As a proof-of-principle, we produced a transgenic mouse line containing Cre-activatable tTA (cl-tTA6). This tTA mouse model was able to induce tumor formation when crossed with a transgenic mouse line containing a doxycycline-inducible oncogene. We also showed that the cl-tTA6 mouse is a valuable tool for faithfully recapitulating the clinical course of tumor development. We showed that FEEST can be easily adapted for other genes by preparing a transgenic mouse model of conditionally activatable EGFR L858R. Thus, FEEST is a technique with the potential to generate transgenic mouse models at a genome-wide scale.
Background Anti-CTLA-4 antibodies have brought about limited clinical benefit because severe toxicity limits dosing levels and/or duration. We used CTLA-4 knockin mice to screen for antibodies with higher anti-tumor activity but lower autoimmunity. We have revealed that the key for better safety and preclinical efficacy is preservation of CTLA-4 for immune tolerance and intratumorial Treg depletion. Our work established that, independent of blocking activities, mAbs that preserve CTLA-4 recycling maintain the physiological immune tolerance checkpoint function while allowing more efficient and selective elimination of tumor-infiltrating regulatory T cells, resulting in highest efficacy and lowest toxicity and was thus developed for clinical testing of all antibodies tested. 1–6 The antibody with best safety and efficacy profile, ONC-392 was developed for clinical testing. The first-in human studies showed that ONC-392 is safe and well tolerated. Remarkably, no irAE has been reported among patients who has received repeated dosing of 3.0 mg/kg and 10.0 mg/kg of ONC-392. The molecular and cellular characterization of ONC-392 will be presented. Methods In vitro binding and disassociation assay were determined between pH 4.0–7.0. The intracellular traffic of both antibodies and CTLA-4 molecules were visualized by confocal microscopy. The binding to human and mouse FcgRI, IIA, IIB, and III (A), FcRn as well as mouse FcgRIV were evaluated by surface plasmon resonance (SPR). Depletion of regulatory T cells in tumor and lymphoid tissues were determined by flow cytometry. Results ONC-392 is a pH-sensitive antibody that preserves CTLA-4 recycling. By preserving cell surface CTLA-4, Onco-392 preserves immune tolerance. Preserving CTLA-4 on tumor-infiltrating Treg contribute to more effective immunotherapy. In addition to its unique feature of pH sensitive binding, OncoC4 also have several important features in Fc. ONC-392 shown comparable binding to human FcgRI and IIIA as wild-type IgG1s. As expected from the mutations introduced, ONC-392 show about 6 fold higher affinity for FcRn than wild-type IgG1. Interestingly, ONC-392 has shown 7–10-fold reduction to FcgRIIB, which is generally considered to be a negative signaling FcR. ONC-392 binding to mouse FcgRI-IV was lower that WT IgG1. Conclusions Unlike other clinical anti-CTLA-4 antibodies, ONC-392 preserves CTLA-4 recycling and thus Treg function in the peripheral tissues. The higher cell surface CTLA-4 allows more efficient Treg depletion in the tumor microenvironment. In addition, despite reduced binding to mouse activating Fc?RI, III/IV, ONC-392 was more effective in intratumor Treg depletion in the mice. Therefore, lacking negative signaling from Fc?RIIB may also contribute to its anti-tumor activity. Trial Registration NCT04140526 References Du X, et al . Uncoupling therapeutic from immunotherapy-related adverse effects for safer andeffective anti-CTLA-4 antibodies in CTLA4 humanized mice. Cell Res 2018; 28 :433–447. Du X, et al . A reappraisal of CTLA-4 checkpoint blockade in cancer immunotherapy. Cell Res 2018; 28 :416–432. Liu Y, Zheng P. How does an anti-CTLA-4 antibody promote cancer immunity? Trends Immunol 2018; 39 :953–956. Zhang Y, et al . Hijacking antibody-induced CTLA-4 lysosomal degradation for safer and more effective cancer immunotherapy. Cell Res 2019; 29 :609–627. Liu Y, Zheng P. Preserving the CTLA-4 checkpoint for safer and more effective cancer immunotherapy. Trends Pharmacol Sci 2020; 41 (1):4–12. Zhang P, et al . Mechanism- and immune landscape-based ranking of therapeutic responsiveness of 22 major human cancers to next generation anti-CTLA-4 antibodies. Cancers 2020; 12 :284.
Phagocytosis is one of the earliest cellular functions, developing approximately 2 billion years ago. Although FcR-based phagocytic signaling is well-studied, how it originated from ancient phagocytosis is unknown. Lipid redistribution upregulates a phagocytic program recapitulating FcR-based phagocytosis with complete dependence on Src family kinases, Syk, and phosphoinositide 3-kinases (PI3K). Here we show that in phagocytes, an atypical ITAM sequence in the ancient membrane anchor protein Moesin transduces signal without receptor activation. Plasma membrane deformation created by solid structure binding generates phosphatidylinositol 4,5-bisphosphate (PIP2) accumulation at the contact site, which binds the Moesin FERM domain and relocalizes Syk to the membrane via the ITAM motif. Phylogenic analysis traces this signaling using PI3K and Syk to 0.8 billion years ago, earlier than immune receptor signaling. The proposed general model of solid structure phagocytosis implies a preexisting lipid redistribution-based activation platform collecting intracellular signaling components for the emergence of immune receptors.
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