The relevance of tumor target expression levels on IgA-mediated cytotoxicity in cancer immunotherapy.

Recent advances in cancer immunotherapy, particularly the success of immune checkpoint inhibitors, have reignited interest in targeted monoclonal antibodies for immunotherapy. Antibody therapies aim to minimize on-target, off-tumor toxicity by targeting antigens overexpressed on tumor cells but not on healthy cells. Despite considerable efforts, some therapeutic antibodies have been linked to dose-limiting side effects. Our hypothesis suggests that the efficacy of IgG leads to a lower target expression threshold for tumor cell killing, contributing to these side effects. Earlier, therapeutic IgG antibodies were reformatted into the IgA isotype. Unlike IgG, which primarily engages Fc gamma receptors (FcγR) to induce antibody-dependent cellular cytotoxicity (ADCC) by NK cells and antibody-dependent cellular phagocytosis (ADCP) by monocytes/macrophages, IgA antibodies activate neutrophils through the Fc alpha receptor I (CD89, FcαRI). In previous studies, it appeared that IgA may require a higher target expression threshold for effective killing, and we aimed to investigate this in our current study. Moreover, we investigated how blocking the myeloid checkpoint CD47/SIRPα axis affect the target expression threshold. Using a tetracycline-inducible expression system, we regulated target expression in different cell lines. Our findings from ADCC assays indicate that IgA-mediated PMN ADCC requires a higher antigen expression level than IgG-mediated PBMC ADCC. Furthermore, blocking CD47 enhanced IgA-mediated ADCC, lowering the antigen threshold. Validated in two in vivo models, our results show that IgA significantly reduces tumor growth in high-antigen-expressing tumors without affecting low-antigen-expressing healthy tissues. This suggests IgA-based immunotherapy could potentially minimize on-target, off-tumor side effects, improving treatment efficacy and patient safety.

The Phospholipid Flippase ATP8B1 is Involved in the Pathogenesis of Ulcerative Colitis via Establishment of Intestinal Barrier Function.

AIMS: Patients with mutations in ATP8B1 develop progressive familial intrahepatic cholestasis type 1 [PFIC1], a severe liver disease that requires life-saving liver transplantation. PFIC1 patients also present with gastrointestinal problems, including intestinal inflammation and diarrhoea, which are aggravated after liver transplantation. Here we investigate the intestinal function of ATP8B1 in relation to inflammatory bowel diseases. METHODS: ATP8B1 expression was investigated in intestinal samples of patients with Crohn's disease [CD] or ulcerative colitis [UC] as well as in murine models of intestinal inflammation. Colitis was induced in ATP8B1-deficient mice with dextran sodium sulphate [DSS] and intestinal permeability was investigated. Epithelial barrier function was assessed in ATP8B1 knockdown Caco2-BBE cells. Co-immunoprecipitation experiments were performed in Caco2-BBE cells overexpressing ATP8B1-eGFP. Expression and localization of ATP8B1 and tight junction proteins were investigated in cells and in biopsies of UC and PFIC1 patients. RESULTS: ATP8B1 expression was decreased in UC and DSS-treated mice, and was associated with a decreased tight junctional pathway transcriptional programme. ATP8B1-deficient mice were extremely sensitive to DSS-induced colitis, as evidenced by increased intestinal barrier leakage. ATP8B1 knockdown cells showed delayed barrier establishment that affected Claudin-4 [CLDN4] levels and localization. CLDN4 immunohistochemistry showed a tight junctional staining in control tissue, whereas in UC and intestinal PFIC1 samples, CLDN4 was not properly localized. CONCLUSION: ATP8B1 is important in the establishment of the intestinal barrier. Downregulation of ATP8B1 levels in UC, and subsequent altered localization of tight junctional proteins, including CLDN4, might therefore be an important mechanism in UC pathophysiology.

ATP8B1 Deficiency Results in Elevated Mitochondrial Phosphatidylethanolamine Levels and Increased Mitochondrial Oxidative Phosphorylation in Human Hepatoma Cells.

ATP8B1 is a phospholipid flippase that is deficient in patients with progressive familial intrahepatic cholestasis type 1 (PFIC1). PFIC1 patients suffer from severe liver disease but also present with dyslipidemia, including low plasma cholesterol, of yet unknown etiology. Here we show that ATP8B1 knockdown in HepG2 cells leads to a strong increase in the mitochondrial oxidative phosphorylation (OXPHOS) without a change in glycolysis. The enhanced OXPHOS coincides with elevated low-density lipoprotein receptor protein and increased mitochondrial fragmentation and phosphatidylethanolamine levels. Furthermore, expression of phosphatidylethanolamine N-methyltransferase, an enzyme that catalyzes the conversion of mitochondrial-derived phosphatidylethanolamine to phosphatidylcholine, was reduced in ATP8B1 knockdown cells. We conclude that ATP8B1 deficiency results in elevated mitochondrial PE levels that stimulate mitochondrial OXPHOS. The increased OXPHOS leads to elevated LDLR levels, which provides a possible explanation for the reduced plasma cholesterol levels in PFIC1 disease.

The phospholipid flippase ATP8B1 is required for lysosomal fusion in macrophages.

ATP8B1 is a phospholipid flippase and member of the type 4 subfamily of P-type ATPases (P4-ATPase) subfamily. P4-ATPases catalyze the translocation of phospholipids across biological membranes, ensuring proper membrane asymmetry, which is crucial for membrane protein targeting and activity, vesicle biogenesis, and barrier function. Here we have investigated the role of ATP8B1 in the endolysosomal pathway in macrophages. Depletion of ATP8B1 led to delayed degradation of content in the phagocytic pathway and in overacidification of the endolysosomal system. Furthermore, ATP8B1 knockdown cells exhibited large multivesicular bodies filled with intraluminal vesicles. Similar phenotypes were observed in CRISPR-generated ATP8B1 knockout cells. Importantly, induction of autophagy led to accumulation of autophagosomes in ATP8B1 knockdown cells. Collectively, our results support a novel role for ATP8B1 in lysosomal fusion in macrophages, a process crucial in the terminal phase of endolysosomal degradation.