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The recognition that after transplantation resident bone marrow-derived cells migrate out of the graft into the recipient has led to an increasing acceptance that the consequent establishment of chimerism is a seminal event in whole organ allograft acceptance, and the first step towards subsequent induction of donor-specific nonreactivity.1–6 Because the migratory interstitial cells are of hematolymphoid origin, the next logical attempt was to augment this natural phenomenon by infusing donor bone marrow cells at the time of whole organ transplantation.
The goal of this study was to induce a higher level of chimerism in unmodified recipients of liver, kidneys, heart, and pancreatic islets by perioperative infusion of unaltered bone marrow from the same donor, in an attempt to either enhance graft survival and/or decrease or ultimately eliminate the requirement for chronic nonspecific immunosuppression.
Sixteen patients were simultaneously transplanted with donor bone marrow and various other organs (Table 1). Immediately following whole organ transplantation, and Without cytoreduction or total lymphoid irradiation (TLI) of the recipient, unmodified donor bone marrow cells (3 × 108/kg body weight) were infused through a central IV line. Additionally, in three type I diabetic patients, donor pancreatic islets (5.7 × 105 – 1.5 × 106 islets/recipient) isolated by a modification of the automated method7 were infused intraportally after graft placement and prior to bone marrow infusion.
All patients were maintained on routine immunosuppression with FK 506 and prednisone. The FK 506 dosage was targeted to plasma trough levels of 1.5 to 2.0 ng/mL in the first postoperative month, and 0.6 to 1.2 ng/mL thereafter and adjustments made as indicated by side effects and/or rejection. Episodes of acute rejections were treated with an in increase in FK 506 or a steroid cycle of solumedrol (1 g tapered to 20 mg), whereas, azathioprine, and OKT3 were added to treat recalcitrant rejection episodes.
The presence of donor cells in the recipients’ peripheral blood mononuclear cells (PBMC) was evaluated weekly in the first month after transplantation and monthly thereafter by the following techniques.
Using donor or recipient-specific fluorescein isothiocyanate (FITC)-conjugated anti-HLA class I or class II monoclonal antibodies (Mab), single color FACS® analysis was performed. Frequency of acquired cells <0.5% was considered below the detection threshold or unquantifiable.
Cytocentrifuge preparation of PBMC obtained from four female recipients of male organs were hybridized with biotinylated Y-chromosome specific probe, which was visualized by FITC-conjugated avidin.
Donor DNA was detected in the recipient’s PBMC by PCR, a procedure described previously.4 Additionally, donor DNA was also quantitated by competitive PCR (cPCR) in four female recipients of male organs by a previously described technique.4
Recipients’ PBMCs were used to monitor their in vitro immune reactivity prior to transplantation and monthly thereafter to mitogens (ConA & PHA), recall antigens (tetanus toxoid), mixed leukocyte reactions (MLR), and cell-mediated lymphocytotoxicity (CML) assays.
The infusion of donor bone marrow cells was uneventful in all patients and all allografts are functioning well. Steroids have been discontinued in five of six liver and three of seven kidney recipients, whereas the remainder of the patients are on a weaning protocol. All patients are currently being maintained on FK 506 (ranging from 2 to 23 mg/d) and the two kidney and pancreatic islet recipients are also receiving additional immunosuppression with azathioprine (75 mg/d).
All patients have evidence of circulating donor cells for up to 16 months after transplantation by either flow cytometry, PCR, or FISH. Only two liver recipients exhibited asymptomatic graft vs. host involvement of the skin, which in one regressed spontaneously, whereas in the other, it required a slight increase in her routine immunosuppression (Table 2). Allografts of 10 of 16 recipients also underwent mild to moderate rejection episodes, which in each case was completely resolved by treating with a gram of steroids followed by a recycle or in one case with an additional course of OKT3.
Nine of the 16 recipients, in whom interpretation of in vitro immune testing was possible, exhibited evolving donor-specific hyporeactivity as early as 50 days after transplantation, which in most cases was sustained for up to the last sample tested (POD 225).
It is too early to predict the impact of donor bone marrow cell augmentation on either allograft survival or on our capacity to wean and/or eventually withdraw immunosuppression. Nevertheless, perioperative infusion of bone marrow was safe, which in each recipient resulted in the establishment of “macrochimerism.” Furthermore, serial in vitro monitoring data suggest that simultaneous infusion of bone marrow along with the whole organ confers an advantage to these patients, for the majority of them exhibit evolving donor-specific hyporeactivity.
Supported by NIH grant DK29961.