Systemic expression of rat arrestin/S-Ag by retroviral gene transfer into hematopoietic stem cells for studies of immune tolerance

John D Cameron, S. W. McPherson, J. P. Roberts, D. S. Gregerson

Research output: Contribution to journalArticlepeer-review

Abstract

Purpose. The role of sequestration in retinal immune privilege remains uncertain. In previous reports, rat S-Ag sequences that we predicted to occupy class II MHC were uveitogenic, consistent with sequestration. Conversely, rat S-Ag is less antigenic and pathogenic than bovine or human S-Ag, arguing for tolerance. The sequence homology between arrestins may allow some tolerance to S-Ag to result from from MHC-restricted presentation of shared, cross-reactive sequences expressed in the thymus. If defects in tolerance to S-Ag result from sequestration, then systemic expression should lead to tolerance. Methods. A mouse fibroblast cell line (GP-SAg) that produces an infectious, replication-deficient, retrovirus carrying the S-Ag gene has been produced by us. Bone marrow (BM) cells from rats (LEW or LEWxBUF-F1's) treated with 5′-FU were isolated by femural perfusion and co-cultured with GP-SAg cells to transduce the stem cells. The transduced stem cells were used to reconstitute lethally irradiated LEW rats. This results in a radiation/BM chimeric rat systemically expressing S-Ag within the context of a "rematuring" immune system. The presence, transcription, and expression of the S-Ag gene are assayed by PCR, RT-PCR, and western blotting, respectively, of peripheral blood lymphocytes (PBL). Tolerance will be assayed by determining susceptibility to experimental autoimmune uveorctinitis (EAU) following immunization, with S-Ag or S-Ag peptides. Results. PCR analysis of PBL's showed the S-Ag gene to be present in 8 of 10 chimeric rats with preliminary evidence for S-Ag transcription in at least one chimeric rat. The presence of the S-Ag gene was detectable for at least five months after reconstitution. Conclusions. The fact that S-Ag can be detected in PBL's indicates that the S-Ag gene has been successfully and stablely introduced into stem cells. We predict that transduction of hematopoietic stem cells by retroviral vectors carrying S-Ag will result in the systemic expression of S-Ag.

Original languageEnglish (US)
Pages (from-to)S540
JournalInvestigative Ophthalmology and Visual Science
Volume37
Issue number3
StatePublished - Feb 15 1996

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