No abstract

Severe hepatic inflammation is a common cause of acute or chronic liver disease. Macrophages are one of the key mediators which regulate the progress of hepatic inflammation. Increasing evidence shows that the TAM (TYRO3, AXL and MERTK) family of RTKs (receptor tyrosine kinases), which is expressed in macrophages, alleviates inflammatory responses through a negative feedback loop. However, the functional contribution of each TAM family member to the progression of hepatic inflammation remains elusive. In this study, we explore the role of individual TAM family proteins during autophagy induction and evaluate their contribution to hepatic inflammation. Among the TAM family of RTKs, AXL (AXL receptor tyrosine kinase) only induces autophagy in macrophages after interaction with its ligand, GAS6 (growth arrest specific 6). Based on our results, autophosphorylation of 2 tyrosine residues (Tyr815 and Tyr860) in the cytoplasmic domain of AXL in mice is required for autophagy induction and AXL-mediated autophagy induction is dependent on MAPK (mitogen-activated protein kinase)14 activity. Furthermore, induction of AXL-mediated autophagy prevents CASP1 (caspase 1)-dependent IL1B (interleukin 1, β) and IL18 (interleukin 18) maturation by inhibiting NLRP3 (NLR family, pyrin domain containing 3) inflammasome activation. In agreement with these observations, axl mice show more severe symptoms than do wild-type (Axl) mice following acute hepatic injury induced by administration of lipopolysaccharide (LPS) or carbon tetrachloride (CCl). Hence, GAS6-AXL signaling-mediated autophagy induction in murine macrophages ameliorates hepatic inflammatory responses by inhibiting NLRP3 inflammasome activation.

Lymphocyte activation gene-3 (LAG-3; CD223), a structural homolog of CD4, binds to MHC class II molecules. Recent research indicated that signaling mediated by LAG-3 inhibits T cell proliferation, and LAG-3 serves as a key surface molecule for the function of regulatory T cells. Previous reports demonstrated that the majority of LAG-3 is retained in the intracellular compartments and is rapidly translocated to the cell surface upon stimulation. However, the mechanism by which LAG-3 translocates to the cell surface was unclear. In this study, we examined the trafficking of human LAG-3 under unstimulated as well as stimulated conditions of T cells. Under the unstimulated condition, the majority of LAG-3 did not reach the cell surface, but rather degraded within the lysosomal compartments. After stimulation, the majority of LAG-3 translocated to the cell surface without degradation in the lysosomal compartments. Results indicated that the cytoplasmic domain without Glu-Pro repetitive sequence is critical for the translocation of LAG-3 from lysosomal compartments to the cell surface. Moreover, protein kinase C signaling leads to the translocation of LAG-3 to the cell surface. However, two potential serine phosphorylation sites from the LAG-3 cytoplasmic domain are not involved in the translocation of LAG-3. These results clearly indicate that LAG-3 trafficking from lysosomal compartments to the cell surface is dependent on the cytoplasmic domain through protein kinase C signaling in activated T cells.

As a potent lymphocyte activator, interleukin-2 (IL-2) is an FDA-approved treatment for multiple metastatic cancers. However, its clinical use is limited by short half-life, low potency, and severe in vivo toxicity. Current IL-2 engineering strategies exhibit evidence of peripheral cytotoxicity. Here, we address these issues by engineering an IL-2 prodrug (ProIL2). We mask the activity of a CD8 T cell-preferential IL-2 mutein/Fc fusion protein with IL2 receptor beta linked to a tumor-associated protease substrate. ProIL2 restores activity after cleavage by tumor-associated enzymes, and preferentially activates inside tumors, where it expands antigen-specific CD8 T cells. This significantly reduces IL-2 toxicity and mortality without compromising antitumor efficacy. ProIL2 also overcomes resistance of cancers to immune checkpoint blockade. Lastly, neoadjuvant ProIL2 treatment can eliminate metastatic cancer through an abscopal effect. Taken together, our approach presents an effective tumor targeting therapy with reduced toxicity.

Bispecific T-cell engagers (BiTEs) that preferentially target tumor-associated antigens (TAA) to reengage CD3 signaling have been approved to treat acute B-cell lymphoblastic leukemia. However, their applications in solid tumors have been hampered due to short half-life, weak antitumor activity, and severe toxicity at therapeutic doses. To explore new targets, we designed a bispecific antibody (BsAb) which simultaneously targets CD3 and immune checkpoint PD-L1. Compared with conventional TAA based targeting, PDL1xCD3 generates far superior anti-tumor immune responses in vivo. Mechanistically, blockade of PD-L1 on dendritic cells instead of tumor cells can potently rejuvenate preexisting tumor reactive CD8 T cells in a B7-1/2 dependent manner for a durable anti-tumor responses. This study argues that targeting DC-T cell instead of current tumor-T cell can achieve much better T cell rejuvenation in BsAb therapy.