APG101 protects immune cells from activation-induced cell death by blocking pro-apoptotic CD95/CD95L signaling

• Published in: European journal of cancer (1990) Vol. 69; pp. S98 - S99
• Main Authors: Merz, C, Sykora, J, Richards, D.M, Fricke, H, Gieffers, C
• Format: Journal Article
• Language: English
• Published: Oxford Elsevier Science Ltd 01.12.2016
• Subjects: Antibodies; Antigens; Apoptosis; Biotechnology; Blood vessels; CD28 antigen; CD3 antigen; CD95 antigen; Cell activation; Cell culture; Cell death; Chemical attack; CTLA-4 protein; Cytometry; Deactivation; Exposure; FasL protein; Flow cytometry; Hematology, Oncology and Palliative Medicine; Homeostasis; Immune checkpoint; Immune system; Immunosurveillance; Immunotherapy; Inactivation; Ligands; Lymphocytes; Lymphocytes T; Macrophages; Monocytes; Mortality; PD-1 protein; Receptors; Signaling; Tumor cell lines; Tumors
• ISSN: 0959-8049; 1879-0852
• DOI: 10.1016/S0959-8049(16)32892-1

Background: Inhibitory receptors on immune cells like PD-1 or CTLA-4 -now termed immune checkpoints - are used by cancers expressing cognate checkpoint ligands in order to escape immune surveillance. The classical death receptor CD95 is a regulator of immune homeostasis and is consequently upregulated following activation of immune cells. Initiation of apoptosis in activated immune cells upon contact with CD95 ligand (CD95L) makes CD95 another putative immune checkpoint. Like other checkpoint ligands, the CD95 ligand is frequently overexpressed in cancers and tumor-associated blood vessels, which possibly facilitates killing or inactivation of immune cells (i.e., tumor counter-attack). Here we examined the mode-of-action of APG101, a drug designed to neutralize CD95L, in protection of immune cells from activation induced cell death and subsequent effects on tumor cell killing. Methods: Monocytes isolated from healthy-donor blood samples were differentiated in vitro into either M1- or M2-type macrophages, which was confirmed by multicolor-flow cytometry for subtype markers. Subsequently, we analyzed the respective M1 and M2-type macrophages regarding apoptosis induction upon CD95L exposure. Analogous experiments were performed using purified T-cells. Analytical FACS was used to monitor apoptosis by appearance of cleaved PARR Real-time cell analysis was performed on direct co-cultures of activated T-Lymphocytes and monocyte-depleted PBMCs with tumor cell lines to monitor tumor cell killing by immune cells. Results: Differentiated M1- and M2-type macrophages express specific marker antigens including CD95. Exposure of macrophages to soluble recombinant CD95L induces apoptosis in both populations. Higher sensitivity of M1- compared to M2-macrophages is observed, coinciding with higher expression of CD95 on M1-macrophages. Addition of APG101 dose-dependently protects macrophages from CD95L-induced cell death. Similar results are obtained after treatment of T-cells with CD95L and APG101 in apoptosis-assays. In direct co-culture, purified T-Lymphocytes and monocyte-depleted PBMCs are able to induce apoptosis in several tumor cell lines. Exogenously added APG101 does not interfere with chemical-induced activation or stimulation of immune cells by anti-CD3 and -CD28 antibodies and, importantly, tumor cell killing is not impaired. Conclusion: APG101 is a potent inhibitor of pro-apoptotic CD95/CD95L signaling and protects activated immune cells from AICD by undergoing apoptosis through exposure to CD95 ligand. Importantly, APG101 does not interfere with macrophage differentiation or activation of the cytolytic functions of T-Lymphocytes and PBMCs in vitro.