Background: We have previously demonstrated that immunotherapy with donor derived Natural Killer (NK) cells has a potential for preventing HCC recurrence in living LT patients (J Clin Invest 2009). We next hypothesized that augmenting phagocytic activity of macrophages, which also play crucial roles in innate immunity against neoplastic cells, might exhibit anti-HCC activity. Signal regulatory protein (SIRP) Α is a critical immune inhibitory receptor on macrophages, and its interaction with CD47 on cancer cells, a ligand for SIRPΑ, likely prevents phagocytosis. In the current study, we investigated whether blocking CD47-SIRPΑ signaling using anti-SIRPΑ mAbs allowed for increased phagocytosis of HCC by macrophages.

Methods: On in vitro phagocytosis assay, Hepa1-6 mouse HCC target cells were labeled with carboxyfluorescein succinimidyl ester (CFSE) and incubated with syngeneic B6 peritoneal cavity (PerC) macrophages together with either anti-mouse SIRPΑ blocking mAbs or isotype-matched control Abs. Macrophages counterstained with allophucocyanin-conjugated anti-mouse F4/80 and phagocytosis of CFSE-labeled targets were measured by FCM analysis in the presence or absence of methylprednisolone. On in vivo phagocytosis assay, CFSE-labeled Hepa1-6 cells were injected into B6 mouse PerC. CD47 knock down (KD) was performed using shRNA with lentiviral particles on Hepa1-6 cells. This yielded cells with 70% reduced CD47 expression was confirmed and subjected to both of in vitro and in vivo phagocytosis assays.

Results: On in vitro phagocytosis assay, adding SIRPΑ mAbs enhanced the phagocytic activity of PerC macrophages against Hepa1-6 compared with isotype-matched Ab treatment (p=0.004). Such significantly promoted phagocytic activity with SIRPΑ mAbs was also seen on in vivo phagocytosis assay. CD47 KD Hepa1-6 cells were significantly more sensitive toward macrophage phagocytosis than parental Hepa1-6 cells on in vitro and in vivo phagocytosis assays (p=0.01), consistent with a role for CD47-SIRPΑ interaction in restricting tumor cell killing. This enhanced phagocytic activity induced by adding SIRPΑ mAbs was still retained even in the presence of clinical dose of methylprednisolone.

Conclusions: Thus, our results suggest that blocking the CD47-SIRPΑ signaling is a novel paradigm for the prevention of HCC recurrence in LT recipients as an adjuvant therapy even under steroid induced immunosuppression, which has proven to hamper NK cell function.

« Back to 2013 American Transplant Congress