Direct immunosuppressive effects of EBV-encoded latent membrane protein 1

In neoplastic cells of EBV-positive lymphoid malignancies latent membrane p rotein (LMP1) is expressed. Because no adequate cellular immune response ca n be detected against LMP1, we investigated whether LMP1 had a direct effec t on T lymphocyte activation. In this study we show that nanogram amounts o f purified recombinant LMP1 (rLMP1) strongly suppresses activation of T cel ls. By sequence alignment two sequences (LALLFWL and LLLLAL) in the first t ransmembrane domain of LMP1 were identified showing strong homology to the immunosuppressive domain (LDLLFL) of the retrovirus-encoded transmembrane p rotein p15E. The effects of rLMP1 and LMP1-derived peptides were tested in T cell proliferation and NK cytotoxicity assays and an Ag-induced IFN-gamma release enzyme-linked immunospot assay. LMP1 derived LALLFWL peptides show ed strong inhibition of T cell proliferation and NK cytotoxicity, while ace tylated LALLFWL peptides had an even stronger effect, In addition, Ag-speci fic IFN-gamma release was severely inhibited. To exert immunosuppressive ef fects in vivo, LMP1 has to be excreted from the cells. Indeed, LMP1 was det ected in supernatant of EBV-positive B cell lines (LCL), and differential c entrifugation in combination with Western blot analysis of the pellets indi cated that LMP1 is probably secreted by LCL in the form of exosomes, The am ount of secreted LMP1 in B cell cultures is well below the immunosuppressiv e level observed with rLMP1. Our results demonstrate direct immunosuppressi ve properties of LMP1 (fragments) and suggest that EBV-positive tumor cells may actively secrete LMP1 and thus mediate immunosuppressive effects on tu mor-infiltrating lymphocytes, Moreover, we demonstrate, for the first time, that transmembrane protein-mediated immunosuppression is not solely restri cted to RNA tumor viruses, but can also be found in DNA tumor viruses.