Macrophage Depletion Can Enhance Combined Radiation and Anti-CD40 Monoclonal Antibody Therapy of B-Cell Lymphoma.

Treatment of B-cell lymphoma with combined radiation and anti-CD40 monoclonal antibody (mAb) can result in long-term CD8⁺ T-cell mediated tumor protection. Survival correlates closely with radiation dose, such that for combinations of 5 Gy and anti-CD40 mAb over 80% of animals survive beyond 100 days, but at 2 Gy maximum survival time is only a week over controls. We hypothesized that this reflected a greater initial cell kill with the higher dose, thus de-bulking tumor and slowing down disease progression, and the fact that there is a larger amount of dying tumor cells available for uptake by antigen presenting cells (APC) thus providing a greater source of antigen for subsequent T-cell priming. However, the fate of irradiated tumor cells (ITCs) in vivo following combination therapy, and the precise interaction and influence of host immune response to tumor remains unknown. It is likely that the nature of the APC involved in the clearance of ITCs may vary depending on the amount of cell death occurring and that this in turn may impact upon the quality of the ensuing immune response. An increased understanding of these events would enable the design of strategies to optimise host immune responses after anti-cancer therapies. In an attempt to address some of these issues, we have examined the influence of macrophages (Mø), one of the primary antigen presenting cell populations, on therapeutic outcome following combined radiation and anti-CD40 therapy. Using syngeneic murine B-cell lymphoma models, we have shown that tumor cells undergo apoptosis (as assessed by flow cytometry measuring DNA content with propidium iodide) and importantly, express surface phosphatidylserine (as measured by Annexin V binding) in a radiation dose-dependent fashion. Microscopy using fluorescently labeled tumor cells reveals that most ITCs dying in vivo are taken up by Mø, with the degree of phagocytosis again correlating with radiation-dose and amount of cell death (>40% clearance post-2Gy; >70% clearance post 5Gy). Using clodronate-liposomes, we have been able to deplete Mø in a manner that is both selective and complete (>95% depletion in spleen [primary site of tumor growth] and peritoneum). Following depletion of Mø, clearance of ITCs in vivo is reduced with <20% uptake post 5Gy. To examine the role of Mø on therapeutic outcome we have conducted combination therapies in mice given clodronate-liposomes or control, PBS-liposomes. Whilst the degree of survival after 5 Gy irradiation plus anti-CD40 mAb was unchanged in Mø depleted animals compared to controls, therapy involving low-dose radiation (2Gy) was greatly enhanced in Mø depleted cohorts, with a median survival time of >50 days compared to less than 5 days in control cohorts. In conclusion, depleting Mø and thus reducing the clearance of ITCs in vivo results in enhanced survival after combination therapy with low-dose radiation plus anti-CD40 mAb in lymphoma. We suggest that Mø depletion increases availability of ITCs for uptake and presentation by other APC (eg. dendritic cells), which may promote T-cell responses more effectively and improve therapeutic results and that these new insights can be readily translated to the clinic.