Characteristics of MPL and PPL lymphoma vaccines and immune responses induced following vaccination. (A) Freeze fracture electron microscopic image of membrane proteoliposome (MPL) demonstrates a “bumpy” texture (arrows) resembling that of tumor cell membranes and sections with a ripple texture characteristic of DMPC bilayers. The purified protein proteoliposome (PPL) shows a predominant DMPC ripple texture with bulges that may be caused by protein and associated water pockets trapped between the lamellae. Samples without cryoprotectant were placed between copper specimen carriers and rapidly plunged into liquid propane. Fracturing and replicating was performed at −115°C in a Balzers BAF 400T Freeze-Etch unit (Liechtenstein). Cleaned replicas were viewed on a Philips EM 300 transmission electron microscope (Netherlands) at a magnification of approximately 55 000 fold and photographed using built-in plates. (B) Immunofluorescent staining of membrane proteoliposomes (MPL) with anti-IL-2 PE and anti-mouse IgM PE antibodies demonstrated the presence of IL-2 (bottom left panel) and tumor-derived idiotype (bottom right panel) on the surface of the liposomes. Phase contrast images are also shown for comparison (top panels). Samples consisting of wet mounts in phosphate buffered saline were viewed using a Nikon Optiphot II Epifluorescence microscrope (Melville, NY) at 500 fold magnification with a 40×/0.75 Plan Fluor Ph2 DLL objective (Nikon, Melville, NY). Images were collected using a Spot RT Color Digital camera (Diagnostic Instrumentations, Inc., Sterling Heights, MI) and Image-Pro-Plus #4 acquisition software (Media Cybernetics, Bethesda, MD). (C) Relative expression levels of membrane proteins were measured on 38C13 murine lymphoma cells and on MPL proteoliposomes prepared from a membrane extract of the same cells. Cells and MPL proteoliposomes were incubated with biotin-labeled monoclonal antibodies to the indicated surface proteins, washed, and then incubated with europium (Eu)-labeled streptavidin. Following washing, the amount of bound Eu was measured on a Delfia 1232 time-resolved fluorometer (Wallac, Gaithersburg, MD). The relative expression level of each membrane protein was determined by dividing its Eu value by that of the 38C13 IgM measured in the same preparation (38C13 cells or MPL proteoliposomes; ie the IgM was assigned a relative expression value of 1.0). (D-F) Mice were inoculated once intraperitoneally with MPL vaccine, PPL vaccine, or Id-KLH + GM-CSF and tested for immune responses after 14 days. Draining lymph node cells were cultured in the presence or absence of tumor-specific 38C13 idiotype or control idiotype and proliferation assay (d) and cytokine induction assay for IFN-γ production (E) were performed as previously described.¹¹  Plasma antibody levels specific for 38C13 idiotype were determined by ELISA (F).¹¹  Data in panels d-f are representative of those obtained in 3 independent experiments each.