Xenopus NK cells identified by novel monoclonal antibodies

Early-thymectomized (Tx) Xenopus frogs, which are permanently deficient in T cells, are used as a model sytem for the characterization of novel monocl onal antibodies (mAb) which identify candidate NK cells at the amphibian le vel of evolution. Hybridomas, generated from mice immunized with splenocyte s from Tx Xenopus following B cell and thrombocyte depletion, were screened by flow cytometry. Three mAb (1F8, 4D4 and 1G5) were identified that stain ed increased proportions of splenocytes from Tx compared with control frogs . These mAb identified lymphoid populations from Xenopus spleen, liver and gut which, after 48 h culture in growth factor-rich medium, exhibited spont anous killing of MHC-deficient allotumor targets,mab-defined splenocytes al so rapidly induced apoptosis of such tumor targets. Dual color analysis con firmed that NK cells are neither T nor B cells. Cytospins of splenocytes is olated with anti-NK mAb revealed large lymphoid cells with distinct pseudop odia. Immunohistology indicated each anti-NK mAb routinely labeled cells wi thin the gut epithelium but NK cells were difficult to visualize in spleen sections. Western blotting of spleen, liver and intestinal lysates subjecte d to sodium dodecyl sulfate polyacrylamide gel electrophoresis showed that 1G5 reacted strongly with protein bands of similar to 70-85 kDa, whereas mA b 1F8 and 4D4 stained less intensely, but identified similar protein bands.