Apoptotic bodies represent an optimal source of tumor antigens that can be internalized and presented by DCs for the development of anticancer vaccination. We have investigated 1 ) a method that could efficiently induce apoptosis of NHL cells, 2) the internalization of apoptotic bodies into DCs, and 3) the presentation by DCs of a model antigen expressed in NHL cells. Apoptotic inducing activity of viral infection was tested and compared with staurosporine treatment, a widely used apoptosis inducing agent. The rationale of this novel approch was aimed at inducing an helper immune response by viral antigens. Apoptosis was evaluated by FACs analysis after staining with annexin V-FITC and propidium iodide (PI). RL-19 NHL cell line, RAP-1 NHL primary cells and EBV-trasformed lymphoblastoid cell line (LCL) were treated with staurosporine (1-5 μM for 4-24 hrs) or incubated either with a modified-vaccinia Ankara virus carrying human tyrosinase (MVA-hTyr) gene (MOI 2-30 for 2 hrs) or with a recombinant MFG adenoviral vector (MOI 10-500 for 2 hrs). Staurosporine (at 5 |-4M for 18 hrs) and MVA infection (at a MOI of 15) induced apoptosis of NHL cells or LCL at similar levels (>80% of cells). Tyrosinase expression was detected in MVA-infected cells undergoing apoptosis by FACs analysis. In contrast, adenoviral infection did not caused apoptosis. NHL cells and LCL, previously stained with PKH2-GL aliphatic green fluorochrome, were induced to undergo apoptosis and then incubated with immature monocyte-derived DCs (1:1 ratio, at 37°C). After 24 hrs and immediately before FACS analysis, PI was added to discriminate between DCs that had actually engulfed apoptotic cells (PKH2-GL+/PI-) and DCs that bound but did not internalize them (PKH2-GL+/PI+). The presence of still viable NHL cells or LCL (PKH2-GL+/PI-) after 24 hrs incubation with DCs was negligible.The percentage of DCs that internalized apoptotic bodies obtained after staurosporine or MVA-hTyr treatment was very high resulting in 70+5% and 62±5% of PKH2-GL+/PI- DCs, respectively. Experiments are in progress to investigate whether HLA-A#0201+ DCs engulfed with MVA-hTyr/NHL apoptotic bodies are able to present human tyrosinase peptides to a tyrosinase-specific HLA-A#0201+ restricted CTL clone. In conclusion, our findings suggest a novel method to promote apoptosis and subsequent phagocytosis by DCs for presentation of tumor antigens to the immune system.
|Issue number||11 PART II|
|Publication status||Published - 2000|
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