Presentation description

Background: Medulloblastoma is the most common malignant brain tumor in children. Immune therapies targeting tumor-associated macrophages (TAMs) can promote tumor cell phagocytosis. However, tumor cells inhibit phagocytosis by TAMs by expressing the cell surface protein CD47. Anti-CD47 antibodies stimulate phagocytosis of medulloblastoma cells by disrupting the "don't eat me" signal sent by the TAM cell surface protein SIRPa when it binds tumor cell CD47. This is called antibody-dependent cell phagocytosis (ADCP). ADCP requires TAM-expressed Fc-gamma receptors (FcgR) to bind to the Fc portion of the anti-CD47 antibody. Two known potent inhibitory regulators of FcgR-mediated ADCP are LILRB4 and CD32b. Recently, our laboratory determined that umbilical cord macrophages phagocytose medulloblastoma cells at higher rates than adult macrophages in response to anti-CD47. LILRB4 and CD32b demonstrated higher expression levels in adult macrophages than in umbilical cord macrophages. Our goal was to investigate whether we can knock down CD32b and LILRB4 in adult macrophages with shRNA, resulting in increased rates of medulloblastoma phagocytosis with anti-CD47.
Methods: We subcloned our shRNA constructs into the pLKO-RFP-shCntrl plasmid. After subcloning, we isolated the DNA plasmid and confirmed by sequencing. Subsequently, shRNA plasmid and lentivirus packaging DNA were transfected into HEK293FT cells to produce the virus. Using lentivirus we transduced human macrophages. After we used FACS to measure the protein expression of CD32b and LILRB4 in successfully transfected macrophages.
Results: Using our shRNA containing lentivirus, the expression of CD32b was suppressed by 52.5%, demonstrating successful knockdown. However, the expression of LILRB4 could not be suppressed using the shRNA-containing lentivirus.
Conclusion: Lentiviral constructs can suppress key negative regulators of FcgR-mediated ADCP. We plan to further optimize our inhibition of these negative regulators and determine if this inhibition can improve anti-CD47 ADCP of medulloblastoma cells.