Donor-specific tolerance by perioperative intrathymic injection of bone marrow cells in the rat cardiac allograft model: use of FK506 can shorten the necessary duration of pretransplant intrathymic conditioning.
BACKGROUND
Many strategies of tolerance induction by intrathymic (IT) injection of donor alloantigens have been reported to date; however, the timing of IT injection is usually 1-3 weeks before transplantation.


METHODS
To apply IT injection to cadaveric organ transplantation, 1 x 10(8) fully allogeneic bone marrow cells (BMC) of Buffalo (BUF; RT1b) rats were intrathymically injected into Wistar Furth (WF; RT1u) rats at the time of BUF cardiac allografting with short-course therapy of antilymphocyte serum (ALS) and FK506 in our experimental model.


RESULTS
Allogeneic IT injection of BUF BMC with ALS and FK506 indefinitely prolonged graft survival (mean survival time > 210 days) in all WF rats. On day 130 after grafting, tolerant WF rats accepted donor BUF skin grafts (> 120 days) but not third-party Lewis skin grafts. In control groups, syngeneic IT injection of WF BMC or intravenous injection of donor BUF BMC in combination with ALS/FK506 therapy failed to induce tolerance. In vivo testing was performed during induction (1 month) or during maintenance (6 months of tolerance. In the mixed lymphocyte reaction (MLR), spleen T cells of tolerant rats at 1 month after grafting displayed hyporesponsiveness after stimulation with donor cells. The addition of interleukin (IL)-2 to MLR culture did not restore T-cell responsiveness. Tolerant rats had a significantly decreased frequency of T cytotoxic cell precursors (fTcp) of 1:4,926, and frequency of IL-2-producing T helper cell precursors (fThp) of 1:23,925, compared with naive rats (1: 2,158 and 1:4,266, respectively). By 6 months after grafting, however, the anti-donor MLR proliferative responses of tolerant rats had been restored to the levels of naive splenic T cells. These tolerant rats displayed restoration of the (fTcp) of 1:2,842 and of the (fThp) of 1:5,630, which were comparable frequencies of naive rats. Suppressor T cells did not contribute in this model. In cardiac grafts of tolerant rats induced by IT injection, expression of both Th1 (interferon-gamma and IL-2) and Th2 (IL-4 and IL-10) cytokines was detected in the early phase; thereafter, expression was completely inhibited, except for interferon-gamma in the chronic phase.


CONCLUSIONS
Perfect donor-specific tolerance was obtained by IT injection of donor BMC at the time of transplantation, while alloimmune responses were maintained at levels similar to those of naive rats.
Ito
A
A
First Department of Surgery, Osaka University Medical School, Suita City, Japan.
Ito
T
T
Kamiike
W
W
Moriguchi
A
A
Ohkawa
A
A
Uchikoshi
F
F
Tanaka
S
S
Nakata
S
S
Matsuda
H
H
D019264:Adoptive Transfer / Q000379:methods*; D000818:Animals; D000961:Antilymphocyte Serum / Q000494:pharmacology; D001854:Bone Marrow Cells; D016207:Cytokines / Q000235:genetics; D006085:Graft Survival / Q000187:drug effects; D016027:Heart Transplantation / Q000276:immunology*; D007166:Immunosuppressive Agents / Q000494:pharmacology; D007201:Indicator Dilution Techniques; D007267:Injections; D007432:Intraoperative Period; D007959:Lymphocyte Culture Test, Mixed; D008297:Male; D008954:Models, Biological; D016133:Polymerase Chain Reaction / Q000379:methods; D011300:Preoperative Care; D012333:RNA, Messenger / Q000032:analysis; D012194:RNA-Directed DNA Polymerase; D051381:Rats; D011915:Rats, Inbred BUF; D011917:Rats, Inbred Lew; D011920:Rats, Inbred WF; D016559:Tacrolimus / Q000494:pharmacology; D013950:Thymus Gland; D013997:Time Factors; D018183:Transplantation Chimera / Q000502:physiology*; D019172:Transplantation Conditioning / Q000379:methods; D014184:Transplantation, Homologous
D000961:Antilymphocyte Serum; D016207:Cytokines; D007166:Immunosuppressive Agents; D012333:RNA, Messenger; D012194:RNA-Directed DNA Polymerase; D016559:Tacrolimus