Increased phagocytosis of CML cells by macrophages following CD47 Knockdown

Abstract

Increased phagocytosis of AML cells by macrophages following CD47 Knockdown. MEGAN SHARP Msc Biomedical Sciences (Immunology Branch) Plymouth University 13 Stangray Avenue, Plymouth, Devon PL4 6PU KRIS JEREMY MSB CBiol PGCHE FHEA Lecturer in Biomedical Science, School of Biomedical and Healthcare Sciences, Room B404a, Plymouth University, PL48AA Acute Myeloid Leukaemia (AML) is a type of leukaemia with upregulated CD47 expression. The ligand CD47’s interaction with its receptor, signal regulatory protein alpha (SIRPα) expressed on macrophages, acts as a ‘don’t eat me’ signal through transduction of immunoreceptor tyrosine-based inhibitory motif (ITIM) domains. CD47 is therefore essential as a ‘marker of self’ on many cells such as red blood cells. Many types of cancer, including AML, have exploited this mechanism and have upregulated CD47 expression to avoid phagocytosis by macrophages. Tumour associated macrophages can also have functional roles in cancer progression and exist as antithetical subtypes, known as M1 and M2. Transfection experiments produced a CD47 knockdown using pan small interfering RNA (siRNA) to deduce the effects on the phagocytosis of AML cell lines by M1 and M2 macrophages. Flow cytometric analysis was used to deduce expression of CD47 on the AML cell lines before and after siRNA knockdown, as well as examine the SIRPα expression on macrophage subtypes. A novel fluorescence based phagocytosis assay was undertaken to measure the differences in phagocytosis between the subsets and knockdown experiments. The CD47 knockdown was effective in increasing the phagocytosis of the AML cell lines. There was also a difference in the phagocytosis dependent on macrophage subtype; a particular increase was identified in the M2 macrophage. Possibly due to the higher expression of the receptor SIRPα found in this phenotype. This research highlights that immunotherapies targeting CD47 and macrophage functionality could prove to be effective in the treatment of AML.