Immunologic mechanisms in tolerance produced in mice with nonradiation-based lymphoablation and donor-specific bone marrow.

Background: These experiments evaluate the mechanisms associated with tolerance in mice treated with sirolimus, antilymphocyte serum (ALS), and donor-specific bone marrow (BM).

Methods: Tolerance to fully MHC-incompatible skin allografts was induced as follows: C57Bl/10 (H2b) recipients received 0.5 mL of rabbit anti-mouse polyclonal ALS on days -1, +2, and +5 relative to B10.A (H2k) donor skin grafting on day 0. Sirolimus was given in a single dose (24 mg/kg intraperitoneally) on day 6. Freshly harvested B10.A (H2k) donor-specific BM was administered at a dose of 25 x 10(6) (ALS/BM25/sirolimus) or 150 x 10(6) (ALS/BM150/sirolimus) cells intravenously on day 7. Skin allograft survival was correlated with the recipient's immunologic status. Recipients were assayed for suppressor cell activity (mixed lymphocyte coculture assays), clonal deletion (T-cell receptor Vbeta11 assay), peripheral and thymic chimerism (flow cytometry and reverse transcriptase polymerase chain reaction), and anergy (response to exogenous interleukin 2).

Results: Mice treated with ALS/BM25/sirolimus showed specifically prolonged but not indefinite allograft survival (median survival time 116 days). Allograft survival correlated with donor-specific clonal deletion and the presence of donor class II mRNA in the recipient's thymus. Mice given the ALS/BM150/sirolimus protocol showed indefinite donor-specific tolerance. Tolerance could not be broken with the administration of high doses of interleukin 2. Splenocytes taken from mice 14 days after tolerance induction inhibited donor-specific and third-party mixed lymphocyte culture proliferation in a dose-dependent fashion. This suppression could be ablated by depleting splenocytes of cells of donor origin before use in coculture. Clonal deletion was detectable 30 days after tolerance induction in mice treated with ALS/BM150/sirolimus and was maintained indefinitely.

Conclusion: Induction of tolerance by ALS, BM, and sirolimus results in a state of donor-specific tolerance, and multilineage chimerism evolves that is permanent and associated with clonal deletion of alloreactive T cells.