Abstract
Abstract: :
Purpose: Fas Ligand contributes to immune privilege by triggering apoptosis of Fas receptor positive infiltrating lymphocytes. However, an alternative function of the transmembrane form of FasL (mFasL) is to stimulate inflammation via activating Fas positive cells that mediate innate immunity (i.e. macrophages, neutrophils). It is the latter function we are attempting to utilize in a novel immunotherapy to eliminate ocular tumors. We hypothesize that an intratumor injection of mFasL microvesicles will induce vigorous inflammation, tumor rejection, and long–term systemic protection from metastases. Methods/Results: Microvesicles were prepared from neuroblastoma (Neuro–2a) cells transfected with either a control (FasL negative) vector or murine membrane–only FasL. Western–blot analysis of the experimental microvesicles revealed high levels of mFasL protein. These microvesicles were co–injected with L5178Y–R lymphoma cells into the anterior chamber of DBA/2 mice. Immunofluorescence staining was performed using neutrophil specific (GR1) and macrophage specific (F4/80) antibodies. Injection of tumor cells with control microvesicles showed minimal neutrophilic infiltrate on day 3, gradually increasing by days 6 and 10. Macrophage infiltration was noted at highest levels on day 3, followed by a decrease to near undetectable levels by days 6 and 10. All mice eventually succumbed to metastatic disease by day 13. Injection of tumor cells with mFasL microvesicles, however, induced massive neutrophilic infiltration by day 3, decreasing slightly on day 6, but then increasing again by day 10. Macrophage infiltration was present on day 3 in the experimental group and was prolonged through day 6. Both immune cell–types were located in the corneal stroma and irido–corneal angle. The inflammatory response resulted in a significant delay in tumor growth and metastatic disease with tumor rejection resulting in phthisis occurring in 30% of the mice. Furthermore, the mice that survived the AC tumor inoculations were systemically protected from a secondary subcutaneous challenge of tumor cells, indicating the presence of a tumor specific adaptive immune response. Conclusions:An intratumor injection of membrane FasL microvesicles induces an early vigorous inflammatory response dominated by the infiltration of neutrophils. These data imply that early inflammation is critical in terminating immune privilege and initiating rejection of ocular tumors.
Keywords: tumors • immunomodulation/immunoregulation • anterior chamber