MODIFICATIONS OF THE CONDITIONING REGIMEN FOR ACHIEVING... : Transplantation (original) (raw)
Although current immunosuppressive therapy for clinical organ transplantation has improved allograft survival, treatment-related complications of chronic drug administration still remain a major limitation to long-term success (1-3). Therefore, the induction of donor-specific transplantation tolerance, which would eliminate the need for chronic immunosuppression, remains a major goal of modern transplantation immunology.
Establishment of mixed allogeneic chimerism has been demonstrated to be an effective means of inducing such tolerance in mice (4-6). We have reported recently that mixed allogeneic chimerism can be induced similarly by a nonmyeloablative preparative regimen in cynomolgus monkeys, and that this chimerism is associated with long-term acceptance of marrow donor-derived kidney allografts (7). The conditioning regimen included perioperative antithymocyte globulin (ATG*), nonmyeloablative whole-body irradiation (WBI, 300 cGy), thymic irradiation (TI, 700 cGy), splenectomy, donor bone marrow (DBM) infusion, and posttransplant cyclosporine therapy (cyclosporine [CsA], discontinued after 4 weeks). Morbidity after conditioning with this regimen was primarily related to the 7- to 14-day period of pancytopenia that resulted from the therapeutic protocol.
To confirm the necessity for each element of the preparative regimen and to further define the parameters consistently associated with tolerance induction, kidney allografts have been transplanted into recipients treated with various modifications of the protocol. We report here that lymphocyte depletion seems to be the best predictor of mixed allogenic chimerism, that donor lymphocyte repopulation of >1.5% is associated with long-term allograft survival, and that all parameters of the original preparative regimen seem to be essential for adequate lymphocyte depletion and organ allograft tolerance.
MATERIALS AND METHODS
Animals. Male cynomolgus monkeys weighing 3-8 kg were used. Recipient and donor combinations were preselected for compatible ABO blood types and mismatched cynomolgus leukocyte major histocompatibility complex antigens. Cynomolgus leukocyte class I antigens were defined serologically, and class II antigenic disparity was determined by in vitro reactivity in one-way mixed lymphocyte reaction. Donor and recipient animals were chosen to ensure that at least one anti-class I monoclonal antibody (mAb) could distinguish donor from recipient for posttransplantation detection of chimerism.
All surgical procedures and postoperative care of animals were carried out in accordance with the National Institutes of Health guidelines for care and use of primates, and were approved by the Massachusetts General Hospital Subcommittee on Animal Research.
Conditioning. The time course of the basic conditioning regimen is detailed in Figure 1. The modifications evaluated in the current studies are summarized in Table 1.
ATG. Horse anti-human thymocyte globulin (ATGAM, Upjohn, Kalamazoo, MI) was administered intravenously at a dosage of 50 mg/kg/day on day -3, -2, and -1, or on day -2, -1, and 0.
CsA. Daily intramuscular injections of CsA in oil, except for recipients M4193 and M3993 that received an intravenous preparation (Sandoz, Basel, Switzerland), were initiated on day 1 at a dosage of 15 mg/kg. The dosage was then tapered over 4 weeks to maintain therapeutic serum levels (>300 ng/ml), and CsA was discontinued at day 28.
Deoxyspergualine (DSG). In two monkeys (Table 2, group F), DSG (kindly provided by Sandoz) was administered at 6 mg/kg/day i.v. from days 0-13.
Irradiation. WBI from 60CO (20-30 cGy/min) was administered either as a single dose on day -6 or as two fractions, day -6 and day -5. Thymic irradiation (TI) was administered as a single 700-cGy dose (100-200 cGy/min) on day -1.
Bone marrow transplantation. Bone marrow was obtained from donor iliac crest and/or vertebral bone by multiple aspirations. These bone marrow cells were filtered through a Fenwal straight type blood set with 80-μ filter (Baxter Healthcare Corp., Deerfield, IL) and infused intravenously and without further manipulation, into the recipient at completion of the kidney transplant procedure on day 0.
Kidney transplantation. Donor and recipient animals were anesthetized with intramuscular ketamine (10 mg/kg) plus valium (0.8 mg/kg), and subsequently maintained with intermittent intravenous ketamine. The donor left kidney and ureter were harvested, taking a small patch of donor aorta for use as a Carrel patch to implant into the recipient aorta. Then the recipient underwent heterotopic kidney transplantation and native nephrectomy as described previously (8).
Splenectomy. Splenectomy initially was added to the regimen in an attempt to decrease the severity of the anticipated postirradiation pancytopenia resulting from the preparative regimen. The necessity of this procedure was evaluated in the “no splenectomy group” (Table 1).
Phenotyping of chimeras. Mixed chimerism was evaluated by flow cytometric analysis of peripheral white blood cells (WBC) as described previously (7). Flow cytometric analysis was performed on a FACScan (Becton Dickinson). Peripheral blood mononuclear cells (PBMCs) were isolated by standard water shock treatment to remove contaminating red blood cells. PBMCs were stained with donor-specific mAbs chosen from a panel of mouse anti-human HLA class I mAbs that cross-react with cynomolgus monkeys. PBMCs were incubated for 30 min at 4°C and then washed twice. Cell-bound mAb was detected with fluorescein isothiocyanate-conjugated goat anti-mouse immunoglobulin (Ig) G2a mAb (Biosource, Camarillo, CA), which was incubated for 30 min at 4°C in the dark. In all experiments, the percentage of cells staining with each mAb was determined from one-color fluorescence histograms and comparison with those obtained from donor and pretreatment frozen recipient cells, which were used as positive and negative controls, respectively. The percentage of cells considered positive was determined using a threshold chosen as the fluorescence level at the beginning of the positive peak, and by subtracting the percentage of cells stained with an isotype control mAb. By using forward and 90° side light scatter (forward scatter [FSC] and side scatter [SSC], respectively) dot plots, lymphocyte (FSC-low and SSC-low), granulocyte (FSC-low and SSC-high), and monocyte (FSC-high but SSC-low) populations were gated, and mixed chimerism was determined separately for each population. Nonviable cells were excluded by propidium iodide staining.
Pathology studies. Allografts were biopsied periodically during periods of stable function, as well as whenever a significant rise in the serum creatinine occurred. Complete autopsies were performed on all euthanized recipients. All tissue was processed for routine light microscopy (hematoxylin and eosin, periodic acid-Schiff stain on kidneys). Frozen sections of selected kidneys were stained for IgG, IgM, C3, and fibrin by standard immunofluorescence techniques.
RESULTS
Table 1 summarizes the various therapeutic modifications studied. Two monkeys were treated with the basic preparative regimen but without DBM. No evidence of chimerism was detected in either recipient, and kidney allografts were rejected on days 51 and 52. Two monkeys treated with the basic preparative regimen but without CsA similarly developed no evidence of chimerism and rejected their kidney allografts on days 11 and 15. Two monkeys treated with the basic preparative regimen but without WBI and TI also did not develop chimerism, and grafts were rejected on days 47 and 61. One of three monkeys treated with the basic preparative regimen but without splenectomy developed chimerism, but unfortunately died of a surgical complication on day 23. Neither of the other two monkeys developed chimerism and rejected on days 30 and 117.
To define the degree of myelosuppression required to allow induction of mixed chimerism and allograft tolerance, we further modified the WBI protocol. Table 2 and Figure 2 summarize the WBI regimens evaluated and their effects on bone marrow suppression, chimerism induction, and allograft survival.
Pancytopenia was observed between days 7 and 20 in all monkey recipients of WBI exceeding 150 cGy. Our initial studies (7) used the single fraction WBI dose (300 cGy) previously found effective in mice (5). With this regimen (group E), one of three monkeys (M393) developed mixed chimerism between days 8 and 34 and subsequently survived more than 800 days without administration of any immunosuppression after day 28. One recipient of this group (M4193) died of sepsis on day 14 during the period of aplasia, and the third monkey (M3993) survived for 175 days, but did not show any evidence of chimerism and experienced a course consistent with chronic rejection by clinical and pathological criteria. As reported previously (7), the latter 2 animals had been treated with an intravenous, rather than intramuscular, CsA preparation, resulting in inadequate serum levels.
In one animal (M3893), the WBI was reduced to 150 cGy. This animal did not develop pancytopenia but demonstrated only transient mixed lymphoid chimerism on day 13. Rejection of this kidney allograft began shortly after cessation of CsA.
Because of the severe pancytopenia that required blood transfusion support in all three monkeys of group E, the WBI subsequently was maintained at 300 cGy but fractionated to 150 cGy on two successive days in six animals (group D). All six of these animals developed mixed chimerism during the first few weeks after transplant. Coincident with the WBI dosage modification, the pancytopenia was less severe, and monkeys regularly recovered from the nadir of marrow suppression without blood transfusion support. Five of these recipients seemed tolerant by mixed lymphocyte reaction, and by the absence of evidence for active rejection on allokidney biopsies. Two of these animals (M3093 and M3293) died on days 196 and 198, respectively, after operative procedures attempting to relieve ureteral obstruction. One monkey (M594) died of urethral obstruction on day 406. One monkey (M6793) developed immune complex glomerulonephritis with crescents and was sacrificed on day 771. One monkey (M1693) has surpassed 935 days with no evidence of rejection and continues to have normal allograft function. The only recipient suffering allograft rejection in group D was M396. Lymphocyte depletion in this animal was similar to that in the other recipients of group D, but maximum donor lymphocyte chimerism never exceeded 0.4% (Table 3).
To determine whether a regimen with comparable efficacy but reduced morbidity could be defined, we reduced the WBI to a fractionated dose of 125 cGy (group C). Two of the four monkeys that received this modified regimen developed multilineage chimerism. One monkey (M1095) survived long-term, dying of ureteral obstruction at day 155. In the second monkey (M4295) that developed chimerism with this protocol, the proportion of donor lymphocytes detected after the marrow infusion never exceeded 0.1% (Table 3). The allograft in this recipient was rejected on day 50. The other two monkeys in this group did not develop chimerism and died secondary to rejection or a ureteral complication.
As summarized in Table 2, the peripheral WBC counts were decreased significantly between days 9 and 22 in all irradiated monkeys, and the degree of WBC depletion was proportional to the WBI dose administered. The WBC counts were suppressed only transiently in the monkeys treated with a fractionated dose of 125 cGy (group C), and the nadir never fell below 1500/mm3. In the monkeys treated with fractionated doses of 150 cGy (group D), the WBC counts were consistently depressed to less than 1000/mm3. In all three monkeys treated with a single dose of 300 cGy (group E), the WBC counts were suppressed to less than 100/mm3. As shown in Figure 2, the peripheral blood lymphocyte population was similarly depleted between days 0 and 18 in all groups except for group A. The severity and duration of the lymphopenic interval was similarly proportional to the intensity of the WBI regimen administered. The initial lymphocyte depletion apparently resulted from the combined effects of irradiation and ATG administration. The lymphopenia was therefore less profound in group A. As summarized in Figure 2, the lymphocyte counts recovered rapidly to above 200/mm3 in group C, whereas in groups D and E, depression to less than 150/mm3 between days 7 and 14 persisted in all animals.
In an attempt to determine whether effective conditioning could be achieved with a combination of the lower WBI dose and pharmacologic manipulation, a 2-week course of DSG was added to the regimen (group F). Both monkeys treated with this modified regimen had multilineage chimerism repeatedly detected. One of these monkeys (M1795) died of a ureteral complication on day 42. At this point, peripheral blood multilineage chimerism remained detectable (granulocyte, 8.5%; monocyte, 1%). Histopathology revealed no evidence of rejection of this allograft. The other monkey (M2895) also developed multilineage chimerism but with maximum donor lymphocyte chimerism of only 0.7% (Table 3). In this recipient the kidney allograft was rejected on day 106. As shown in Table 2, the degree of WBC and lymphocyte depletion was more severe in these recipients than in the monkeys treated with a fractionated dose of 125 cGy but without DSG.
Figure 3 shows the typical pattern of multilineage chimerism detected by flow cytometry in successfully engrafted recipients. In this animal (M1095), the chimerism reached its maximum at about day 20 in all subpopulations, after which the proportion of donor cells gradually declined until day 36, after which it became impossible to distinguish donor cells from background staining.
Table 3 summarizes the correlation between the degree of chimerism detected by flow cytometry and prolongation of graft survival. Multilineage chimerism was detected in all of these recipients except for M6793. (Because of technical limitations in the earlier studies, lymphocyte chimerism was not evaluated before day 23 in this recipient.) In four monkeys, monocyte chimerism could not be analyzed because of the high background. All recipients that achieved lymphocytic chimerism of greater than 1.5%, except for M1795, which was lost to a technical complication, had long-term renal allograft survival without evidence of rejection. In contrast, three recipients (M2895, M4295, and M396) that developed multilineage chimerism but had lymphocyte chimerism of less than 1% proceeded to reject their grafts.
Although long-term kidney allograft survival without evidence of rejection was observed in the effectively conditioned recipients, many of these animals ultimately died of ureteral obstruction (Table 2). The obstructive process was typically found to involve the entire donor ureter except for the proximal renal pelvis. The pathology of the obstructed allograft ureters from some of the animals in this study are summarized in Table 4. Those animals with rejection of the kidney sometimes had a prominent inflammatory infiltrate of mononuclear cells and eosinophils that invaded the mucosa and smooth muscle. The epithelium was often sloughed and the smooth muscle necrotic. Endothelialitis was prominent in submucosal arteries, characterized by subendothelial infiltration of mononuclear cells and eosinophils. No viral inclusions were seen. As shown in Figure 4, in the animals without rejection in the kidney, the ureters often showed some of the same changes, including an infiltrate of mononuclear cells and eosinophils that invaded the epithelium and endothelialitis but without smooth muscle necrosis.
DISCUSSION
In preparation for extension of this approach to clinical trials, we have attempted to refine the protocol to limit potential morbidity. Because lower dosages of WBI are associated with less toxicity, it was important to identify the lowest WBI dose that would permit stable donor bone marrow engraftment. Tomita et al. (9) reported that between 150 and 300 cGy WBI is needed to permit syngeneic pluripotent hematopoietic stem cell (HSC) engraftment in a murine model. We evaluated, therefore, the correlation between the degree of myelosuppression resulting from various dosages of WBI and allograft survival. The single fraction of 300 cGy was unacceptably toxic. All three monkeys receiving this regimen had severe pancytopenia that required life-saving transfusion of blood products. Despite this support, one recipient died of sepsis during the period of leukopenia. The fractionated dose of 125 cGy was less toxic and did not require blood product support in any monkey. However, this modification proved insufficient for consistent and adequate recipient WBC and lymphocyte depletion. After the initial ATG and WBI induced lymphopenia, the lymphocyte counts in these recipients recovered rapidly to above 200/mm3. Only two of these four monkeys developed multilineage chimerism, and long-term tolerance was achieved in only one recipient. In contrast, all six monkeys treated with a fractionated dose of 150 cGy also recovered without need for blood transfusion support, but lymphocyte depletion to less than 150/mm3 persisted for approximately 2 weeks. All but one of these recipients developed multilineage chimerism and long-term survival with donor-specific unresponsiveness.
Previously, Vriesendorf (10) had demonstrated that myelosuppressive treatment is required to create the poorly defined entity known as “space” needed for HSC engraftment. Tomita et al. (9) have suggested that a major role of such myelosuppressive treatment may be to damage and reduce the numbers of host HSCs and that destruction of recipient hematopoietic cells may increase the number of physical “niches” in the marrow microenvironment that are available to injected HCS. Our results demonstrate that lymphocyte depletion seems to be an important predictor of successful induction of mixed chimerism. Thus, lymphocyte depletion seems to be a good marker of a permissive environment known as “space” for bone marrow cell engraftment. Our observations further emphasize that the degree of WBC and lymphocyte depletion are proportional to the WBI dose administered (Table 2, Fig. 2) and suggest that persisting lymphocyte depletion during the first several weeks after transplantation is predictive of successful chimerism induction. In this model, the fractionated dose of 125 cGy proved to be a marginal dose to develop chimerism, whereas the fractionated dose of 150 cGy consistently permitted stable donor bone marrow cell engraftment. In group F, the fractionated dose of 125 cGy with DSG proved to be sufficient for adequate WBC and lymphocyte depletion. The number of recipients treated with DSG will need to be enlarged to determine whether it is effective in this model.
Although a number of investigators have reported successful engraftment of donor organs in the mixed chimeric state (11, 12), it is not clear from those studies what threshold of chimerism is required for the induction of donor-specific tolerance. Recently, Taniguchi et al. (13) reported that recipient mice with >30% chimerism could accept skin grafts from marrow donor mice, whereas those with <10% chimerism showed prolonged but not permanent graft survival.
Our current studies also suggest that successful engraftment of limited proportions of DBM cells may not be sufficient for induction of donor specific allograft tolerance. Three monkeys (M4295, M2895, and M396) rejected allokidneys on days 36, 50, and 106 after transplant, despite having developed multilineage chimerism. The levels of granulocyte and monocyte chimerism in these three monkeys were similar to those achieved in the long-term surviving recipients. However, the maximum proportion of lymphocyte chimerism in these three monkeys was less than 1%, in contrast to that in the long-term surviving monkeys, where maximum lymphocyte chimerism exceeded 1.5% (Table 3). Thus, maximum lymphocyte chimerism of greater than 1.5% seems to be more consistently associated with induction of tolerance. This observation would have obvious clinical implications for patients being considered for withdrawal of CsA therapy after conditioning with this regimen. The discrepancy between the degree of chimerism in our recipients versus those reported by Taniguchi et al. may be explained by several differences in the studies. Most obvious are the recognized differences between rodent and primate models. In addition, we infused only DBM, whereas in the study of Taniguchi et al., DBM mixed with recipient bone marrow was injected into recipients. Finally, the recipient mice were irradiated lethally with a single dose of 950 cGy, whereas our recipients received 250-300 cGy of WBI.
Ureteral stenosis had not been observed in our previous monkeys with shorter-term observation after renal transplantation (8) using other immunosuppressive protocols and has apparently been encountered by other investigators evaluating regimens designed to induce tolerance as well (F. Thomas, personal communication). As shown in Table 2, an unanticipated high incidence of ureteral stenosis in long-term survivors was observed in this study. Although there was no evidence of rejection in the kidney itself, we interpret the ureteral changes as probably a result of rejection of the ureter of varying intensity. Infection or ischemia are the two other possible explanations. The most convincing evidence in favor of rejection is the endothelialitis. The most severe changes (with smooth muscle necrosis) were found primarily in association with rejection of the kidney in short-term survivors (Table 4). However, in three long-term survivors without kidney rejection, severe fibrosis was found that led to obstruction of the ureter, and two of these had focal endothelialitis, arguing that rejection of the ureter was responsible. It is known that the polyoma virus designated BK virus can cause ureteral stenosis in humans (14). Although our data do not exclude this possibility, we were unable to detect viral inclusions in these ureters, which are usually readily demonstrable in clinical samples.
It was unexpected that rejection of the ureter could occur in the absence of rejection of the kidney from the same donor. In the human, although ureteral rejection has been reported (15-18), we are unaware of any documented cases of isolated rejection of the ureter without rejection of the kidney. The reasons for selective tissue rejection could relate to tissue-specific antigens (in analogy to the skin), differences in antigen accessibility or ancillary cofactors, or poor healing of the ureter, in contrast to the kidney after earlier rejection activity. (Early posttransplant biopsies not infrequently reveal interstitial infiltrates that disappear without treatment.)
Several investigators have reported that the incidence of ureteral obstruction after kidney transplantation ranged between 2 and 7.5% (15-18). Keller et al. (19) emphasized that the ureteral stenosis that occurred in about 3% of renal transplants in man typically arises in the distal ureter in the first 3 months after transplantation. Thomalla et al. (20) also demonstrated that the majority of urological complications occurred in the early postoperative period; late occurrences (beyond 3 months) were much less common. This complication is, therefore, usually attributed to distal ischemia. Thomalla (20), therefore, suggested that late ureteral complications may occur as a result of acute or chronic rejection involving the donor ureter and leading to compromised vasculature with resultant ischemia, fibrosis, and stricture formation. On the basis of these observations, it may be necessary to provide urinary drainage, which utilizes the recipient ureter (ureteropyelostomy) to avoid this complication in clinical trials.
Despite this ureteral problem, we believe that this nonmyeloablative preparative regimen is appropriate for extension to clinical trials. We conclude from the studies presented here that all parameters of the original preparative regimen seem essential for adequate lymphocyte depletion and induction of allograft tolerance. In addition, lymphocyte depletion seems to be the best predictor of mixed chimerism, and donor lymphocyte repopulation of at least 1.5% is associated with long-term allograft survival.
Acknowledgments. The authors thank Dr. Siew Lin Wee for advice on performance of in vitro studies; Drs. Svjetlan Boskovic, Han Zhou Hong, and Rod Monroy for technical assistance; and Cathy Padyk for expert assistance in preparing the manuscript.
Non-myeloablative protocol for induction of tolerance in cynomolgus monkeys. Schematic timeline of protocol used for induction of mixed chimerism and tolerance in cynomolgus monkeys is detailed. WBI was administered on day -6 or days -6 and -5. Thymic irradiation was administered on day -1. ATG was administered at 50 mg/kg i.v. on days -3, -2, and -1. Kidney transplantation and splenectomy were performed on day 0. DBM cells were infused on day 0 after completion of kidney transplantation procedure. Daily CsA was administered intramuscularly on days 1-28.
Peripheral blood lymphocyte counts of monkeys receiving different doses of WBI. Lymphocyte counts decreased transiently between days 0 and 20 in all groups, and the degree of lymphocyte depletion was proportional to the WBI dose administered.
M1095 mixed chimerism by flow cytometry. The typical pattern of multilineage chimerism detected by flow cytometry in recipient M1095 after transplantation. Multilineage chimerism reached its maximum around day 20. Thereafter, the proportion of donor cells gradually decreased until it became impossible to distinguish from background staining (after day 36).
Light microscopy of the transplanted kidney and ureter from M393 on day 824. (Top) Allokidney: There is no evidence of rejection. Vessels are normal. Mild focal interstitial fibrosis and tubular dilatation are present with no interstitial mononuclear infiltration. (Bottom) Alloureter: There is severe acute ureteritis with eosinophils especially in submucosa. Smooth muscle and vessels are normal.
Footnotes
This work was supported by NIH grant AI37692 and funds from BioTransplant, Inc., Charlestown, MA.
Abbreviations: ATG, antithymocyte globulin; CsA, cyclosporine; DBM, donor bone marrow; DSG, deoxyspergualine; HSC, hematopoietic stem cell; Ig, immunoglobulin; mAb, monoclonal antibody; PBMC, peripheral blood mononuclear cell; TI, thymic irradiation; WBC, white blood cell; WBI, whole-body irradiation.
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