Effect of systemic expression of sequestered antigen on immunologicat tolerance

S-Ag is a retinal rod protein expressed in the brain and eye, but not in peripheral organs such as spleen or thymus. Immunization of LEW rats with S-Ag or peptides leads to experimental autoimmune uveoretinitis (EAU), in dicating that they are not immunologically tolerant to S-Ag. By expressing S-Ag in hematopoietic stem cells used to reconstitute irradiated rats, we have a model in which the immune system matures in the presence of a normally sequestered antigen. Methods: Bone marrow (BM) was collected from 5'FU-treated donors. Red cells were lysed, and the remaining cells cocultured for 3 days with fibroblasts producing virions carrying S-Ag and <7418r genes. Recipients were lethaily irradiated and reconstituted with BM. After at least 3 months, chimeric status of animals was examined by PCR on blood. Animals were immunized with the 343-362 peptide, the course of EAU followed, and the animals sacrificed. Results: The time course and severity of EAU in chimeric animals was greatly reduced (in most cases non-existent). Responsiveness of draining lymph node cells to immunizing peptide was generally not seen in highly PCR-f chimeras. We were able to grow G4187" spleen cells from the chimeras, and are examining organs for the presence of S-Ag gene, message, and protein. Conclusion: These preliminary results indicate that the systemic expression of a normally-sequestered antigen leads to immunological tolerance to that antigen. Thus, sequestration appears to play a role in the prevention of autoimmunity in the eye. This research was supported by NIH/NEI grants EY09207 (DSG) and a Visual Sciences Training Grant (JPR), and by RPB, Inc.