Detection of regulatory cells as an assay for allograft tolerance in miniature swine.
BACKGROUND
There is currently a great need for an in vitro assay to assess the presence of tolerance following allotransplantation to determine whether immunosuppressive medications can be discontinued. Our laboratory has recently developed an assay involving coculture inhibition of cell-mediated lympholysis that correlates with tolerance to allografts in swine leukocyte antigen (SLA) Class I-mismatched miniature swine. The potential for clinical application of this assay may depend on 2 important factors: (1) whether the assay can be used in the presence of immunosuppression; and (2) whether frozen-stored naive responder cells can be utilized.


METHODS
Long-term tolerant MGH miniature swine that had accepted SLA Class I-mismatched kidney transplants after a 12-day course of cyclosporine or tacrolimus were studied. Two long-term tolerant and 2 naive control animals were treated with a clinically relevant dose of cyclosporine for 2 weeks (trough level 100 to 400 ng/ml) to simulate the ongoing "chronic" immunosuppression used in human recipients of allografts. Cells from tolerant or naive, recipient-matched animals were stimulated for 6 days with donor or third-party SLA. These primed cells were then cocultured with naive unstimulated recipient major histocompatibility complex (MHC)-matched responders and irradiated stimulators. Responder cells were tested both fresh and frozen.


RESULTS
Suppression of cytotoxic responses of naive responder cells was observed in all coculture assays using cells from tolerant animals primed against donor antigen in vitro, but not in assays using similarly primed cells from naive animals. Responder cells from tolerant animals receiving immunosuppression had a suppressive activity similar to that from cells of the same animals not receiving immunosuppression. Similar suppression was also observed in coculture assays using either fresh or frozen naive responder cells.


CONCLUSIONS
This coculture assay appears to correlate with the presence of tolerance under conditions applicable to the clinical setting. The assay appears to identify peripheral regulatory mechanisms of tolerance in allogeneic transplant recipients, and therefore may provide an approach for determining an appropriate timepoint at which to test withdrawal of immunosuppressive medications.
Wu
Anette
A
Transplantation Biology Research Center, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts 02129, USA.
Yamada
Kazuhiko
K
Baron
Christophe
C
Mathes
David W
DW
Monajati
Leila M
LM
Vagefi
Parsia A
PA
Sachs
David H
DH
D000818:Animals; D002478:Cells, Cultured; D018920:Coculture Techniques; D016572:Cyclosporine / Q000627:therapeutic use; D007108:Immune Tolerance; D007165:Immunosuppression Therapy; D007166:Immunosuppressive Agents / Q000627:therapeutic use; D016030:Kidney Transplantation / Q000276:immunology*; D013552:Swine; D013556:Swine, Miniature; D013601:T-Lymphocytes / Q000276:immunology; D014184:Transplantation, Homologous
D007166:Immunosuppressive Agents; D016572:Cyclosporine