Determination of an improved sirolimus (rapamycin)-based regimen for induction of allograft tolerance in mice treated with antilymphocyte serum and donor-specific bone marrow.

BACKGROUND

Posttransplant donor-specific bone marrow (BM) infusion in mice treated with antilymphocyte serum (ALS) induces specific unresponsiveness (tolerance) to skin allografts, which can be augmented by the adjuvant administration of chemotherapeutic immunosuppressive agents. The purpose of this study was to determine the optimal dose and timing of administration of sirolimus (rapamycin) to induce maximal skin allograft survival in ALS-treated, BM-infused recipients.

METHODS

DBA/2 donor skin grafts were placed on B6AF1 recipients (class I- and II-disparate). Groups of recipient mice (n=10 each) received combinations of the following treatment protocols: ALS, 0.5 ml on days -1 and 2; BM, 25x10(6) donor-specific cells on day 7; sirolimus, 6, 12, 18, or 24 mg/kg at times indicated; and cyclosporine, 50 mg/kg at times indicated. The immune status of putatively tolerant animals was examined with mixed lymphocyte cultures, cell-mediated lympholysis assays (CML), and limiting dilution analyses.

RESULTS

When administered in conjunction with ALS/BM, a single dose of sirolimus (6 mg/kg) on days 21, 18, 14, 10, or 7 resulted in median skin graft survival times of 35, 26, 40, 46, and 103 days, respectively, versus a median survival of 27 days in mice given ALS and BM alone. The addition of cyclosporine to sirolimus (6 mg/kg) given on day 7 or days 7 and 10 did not significantly increase graft survival over that achieved with sirolimus alone. A single dose (18 or 24 mg/kg) of sirolimus administered on day 7 to ALS/BM-treated recipients resulted in 100% 200-day skin graft acceptance. Tolerant mice demonstrated nonspecific suppression of the mixed lymphocyte culture assays at 90 and 200 days and a nonspecific reduction of the CML assay at 50 days. By 200 days, the third-party CML response was restored, whereas donor-specific cell-mediated cytotoxicity remained suppressed. There was a donor-specific reduction in the number of alloreactive cytotoxic T lymphocyte clones by limiting dilution assay at 120 days. In vivo specificity of immunosuppression induced with this protocol was demonstrated by indefinite survival of second donor-specific skin grafts placed on putatively tolerant mice at day 90, whereas third-party skin grafts were rejected in 14 days.

CONCLUSION

A single dose of sirolimus (18-24 mg/kg) administered on day 7, within the context of an ALS/BM immunosuppressive regimen, reliably induces permanent skin allograft acceptance in this model. In vitro measures of immunocompetence demonstrated an early nonspecific suppression of the recipients immune status and later recovery of third-party immunoreactivity. In vivo testing indicates an operationally tolerant state that is donor-specific 90 days after treatment.