Induction of stable chimerism and transplantation tolerance to rat islet and heart allografts by ultraviolet-B modulation of bone marrow cells.

Ultraviolet-B irradiation (UV-B) (700 J/m2) of BM cells prior to transplantation into lethally gamma-irradiated (1050 rads) allogeneic rats prevents the development of GVHD and results in stable chimerism. This study was developed to determine if UV-B modulation of BMT is useful for preconditioning recipients for the induction of tolerance to donor islets and heart allografts. Lethally irradiated Lewis rats that received UV-B irradiated (700 J/m2) WF BMT (10(8) BM cells) demonstrated stable chimerism without any evidence of GVHD. The stable Lewis chimeras were made diabetic with streptozotocin (STZ) at 28-35 days after BMT and subdivided into 3 experimental groups that received 1000-1200 islets from WF, Lewis, or BN (third-party), respectively. The results showed that group I diabetic Lewis chimeras accepted permanently (greater than 300 days) BM donor WF islets and became normoglycemic. When 3 of 6 Lewis chimeras transplanted with WF islets were rechallenged with WF hearts 60 days after islet grafts, they accepted both islets and cardiac allografts permanently (greater than 240 days). Similarly, the remaining 3 animals accepted Lewis cardiac allografts permanently, thus indicating tolerance to both donor and recipient alloantigens. Group II diabetic chimeras accepted permanently (greater than 300 days) recipient (Lewis) islets. In contrast, group III chimeras rejected acutely (7-8 days) third-party (BN) islets. However, when these animals that rejected BN islets and again became diabetic were retransplanted with BM donor-type (WF) islets, they became permanently normoglycemic (greater than 200 days). This finding emphasizes the specificity of the induction of tolerance in this model and the apparent lack of organ-specific sensitization. To define the underlying mechanism of tolerance, in vivo adoptive transfer of 10(8) spleen cells to naive Lewis or WF recipients, obtained from tolerant Lewis chimeras carrying donor islets and heart allografts, showed no prolongation of cardiac allografts in the unmodified syngeneic hosts, thus questioning the role of suppressor mechanisms in the tolerant rats. Furthermore, cells from the tolerant chimeras that showed no mixed lymphocyte reaction (MLR) response to Lewis or; WF alloantigens failed to suppress anti-Lewis and anti-WF MLR-response in coculture MLR. These results suggest that tolerance to donor alloantigens in the UV-B BMT model is most likely due to selective elimination of anti-BM donor helper or effector cell precursors (clonal deletion) rather than induction of suppressor cell activity. This study demonstrates that this relatively simple and effective approach to modulation of T cells in BM treatment may be potentially useful in the induction of tolerance to donor organs.     

Short-term immunosuppression facilitates induction of mixed chimerism and tolerance after bone marrow transplantation without cytoreductive conditioning

BACKGROUND: Induction of mixed chimerism and tolerance usually requires cytoreduction or transplantation of high numbers of bone marrow cells (BMC). However, such protocols have only a suboptimal success rate and, more importantly, equivalent numbers of BMC cannot be routinely obtained in the clinical setting. The authors therefore evaluated whether a short-course of immunosuppression (IS) given in addition to co-stimulation blockade would facilitate chimerism induction and allow reduction of the minimally required number of BMC without cytoreduction. METHODS: B6 mice received 200, 100, or 50 x 10 unseparated BMC from Balb/c donors plus an anti-CD40L monoclonal antibody (mAb) and CTLA4Ig (without irradiation or cytotoxic drugs). Some groups were treated additionally with IS (rapamycin, methylprednisolone, and mycophenolate mofetil for 4 weeks after bone marrow transplantation), donor-specific transfusion (DST), or anti-OX40L mAb, as indicated. RESULTS: IS led to long-term multilineage chimerism in 9 of 10 mice receiving 200 x 10 BMC (without IS, 1 of 4; P<0.05), in all mice (n=10) receiving 100 x 10 (without IS, 6 of 9; P<0.05), and notably in 9 of 10 mice treated with 50 x 10 BMC (without IS, 4 of 10; P<0.05). With transient IS, donor skin grafts were accepted longer than 170 days in 9 of 10 mice receiving 200 x 10 (without IS, 0 of 5 mice; P<0.05), all mice receiving 100 x 10 (without IS, 6 of 9; P<0.05), and 6 of 11 mice receiving 50 x 10 BMC (without IS, 4 of 10). The use of DST or anti-OX40L mAb had no beneficial effect. CONCLUSIONS: Transient IS significantly improves rates of chimerism and donor skin graft survival, and allows lasting mixed chimerism after transplantation of only 50 x 10 BMC. Thus, IS might help in the further development of noncytoreductive chimerism protocols.     

Mechanisms of alloimmune tolerance associated with mixed chimerism induced by vascularized bone marrow transplants

Rat limb allograft recipients represent surgically induced, immediately vascularized bone marrow transplant (VBMT) chimeras. The majority of these chimeras undergo tolerance while a minority develop graft versus host disease (GVHD). T-cell chimerism and associated mechanisms of cellular immune nonresponsiveness were investigated in tolerant VBMT chimeras. A strong correlation (p < 0.01) was observed between the clinical onset of GVHD and levels of donor T-cell chimerism approximating or greater than 50%. However, stable mixed chimerism was associated with tolerance. In conclusion, three major sequential mechanisms of immune nonresponsiveness were elucidated in tolerant VBMT chimeras over time and included development of nonspecific suppressor cells (which potentially represent natural suppressor cells), maturation of antigen-specific suppressor cell circuits, and eventually putative clonal inactivation.     

Donor bone marrow transplantation: chimerism and tolerance

Infusion of donor bone marrow (DBM)-derived cells continue to be tested in clinical protocols intended to induce specific immunologic tolerance. Central clonal deletion of donor-specific alloreactive cells associated with mixed chimerism reliably produced long-term graft tolerance. In this setting, depletion of recipient T cells by antilymphocyte antibodies and subsequent repopulation by donor hematopoietic cells after donor bone marrow infusion (DBMI) are prerequisites for tolerance induction. Major advances have been made in animal models and in pilot clinical trials and the key questions with the future perspectives are presented in this article.     

Simultaneous vascularized bone marrow transplantation to promote acceptance of hind limb allografts and its effects on central and peripheral chimerism

Administration of donor bone marrow (BM) cells can improve the outcome of transplantation. The ability of donor vascularized bone marrow transplantation (VBM) to provide an ongoing source of donor cells and improve survival in a rigorous rat model of hind limb transplantation (HLTX) was investigated. HLTX were performed between Brown Norway (BN) donors and Lewis recipients in three groups: HLTX; HLTX plus intravenous donor BM cells and HLTX plus simultaneous VBM transplantation. Animals received 12 weeks triple immunosuppression. Survival was compared at 4 months and donor chimerism was evaluated. Simultaneous VBM transplantation led to slight but nonsignificant prolongation of survival (P=0.056). Donor cells in the VBM were eventually replaced by recipient and there was no long-term increase in chimerism. Few donor cells were observed in thymus. Simultaneous VBM transplantation showed a trend for improved survival of HLTX however the VBM failed to provide a sustained increase in chimerism.     

Evaluation of mixed chimerism in bone marrow transplantation program in Croatia

In this study we monitored mixed chimerism in 36 patients with various hematologic disorders. All of them underwent a classic conditioning regimen, 31 patients for related bone marrow transplantation (BMT) and 5 patients for unrelated BMT. DNA was isolated from peripheral blood, and samples were polymerase chain reaction (PCR) amplified for 5 short tandem repeat (STR) loci (TH01, VWA31, FES/FPS, F13A01, and SE33) and for one variable number of tandem repeats locus (D1S80). Samples were run on a 6% polyacrylamide gel in an automated ALFexpress sequencer. In all 36 donor-recipient pairs we found differences for at least two STR loci. In most cases the difference was observed for SE33 and D1S80 loci. Mixed chimerism (MC) was detected in 18 patients: 4 with unrelated BMT and 14 with related sibling donors. In 11 patients MC was detected in the early period after BMT, but was soon followed by full donor chimerism (FDC) in peripheral blood. In 5 cases patients MC appearing after FDC was established, and was predictive for the relapse. One patient showed alternating MC and FDC, but at the end showed only recipient cells and graft rejection. In conclusion, the PCR-STR analysis is a highly informative, fast, and simple screening method for monitoring chimerism in a BMT program.     

Stable mixed hematopoietic chimerism permits tolerance of vascularized composite allografts across a full major histocompatibility mismatch in swine

This study tested the hypothesis that vascularized composite allografts (VCA) could be accepted in a robust model of hematopoietic chimerism by injecting allogeneic bone marrow cells (BMC) into swine fetuses. Outbred Yorkshire sows and boars were screened to ensure the absence of the major histocompatibility (MHC) allele SLA^cc of inbred MGH miniature swine and then mated. Bone marrow harvested from an SLA^cc swine donor was T‐cell depleted and injected intravenously into the fetuses between days 50–55 of gestation. After birth, the piglets were studied with flow cytometry to detect donor cells and mixed lymphocyte reactions (MLR) and cell‐mediated lympholysis (CML) assays to assess their response to donor. Donor‐matched VCAs from SLA^cc donors were performed on four chimeric and two nonchimeric swine. The results showed donor cell engraftment and multilineage macrochimerism after the in utero transplantation of adult BMC, and chimeric animals were unresponsive to donor antigens in vitro. Both control VCAs were rejected by 21 days and were alloreactive. Chimeric animals accepted the VCAs and never developed antidonor antibodies or alloreactivity to donor. These results confirm that the intravascular, in utero transplantation of adult BMC leads to donor cell chimerism and donor‐specific tolerance of VCAs across a full MHC barrier in this animal model.     

Minimal conditioning required in a murine model of T cell depletion,thymic irradiation and high-dose bone marrow transplantation for the induction of mixed chimerism and tolerance

In a recently developed murine model for the induction of mixed chimerism and tolerance, hosts are treated with T cell depleting monoclonal antibodies (TCD mAbs; days -5, -1 and +7), thymic irradiation (TI) (7 Gy), and a high dose of fully allogeneic bone marrow cells (BMC, 200 x 10(6)). To find the minimum amount of each treatment required for success with this approach, we treated groups with (1) a lower dose of TI (3.5 Gy), (2) fewer BMC (100 x 10(6)), (3) no TI, (4) no TI plus additional TCD mAbs on day +14, or (5) fewer injections of TCD mAbs (day -5 only). Chimerism was followed by flow cytometry (FCM), and tolerance was assessed by skin grafting. Without TI, no long-term chimerism or tolerance could be induced, even when an additional dose of TCD mAbs was administered on day +14. A reduction in the dose of either BMC or TI led to substantially reduced effectiveness, as demonstrated by lower levels of chimerism and poorer donor skin graft survival. However, the dose of TCD mAbs and hence the duration of recipient T cell depletion could be safely reduced and thus the potential toxicity of the conditioning regimen lowered.     

Persistence of stable intragraft cell chimerism in rat liver allografts after drug-induced tolerance.

BACKGROUND: Drug-induced tolerance of rat liver allografts is well documented. We analyzed cellular events during immunosuppressive therapy on day (d) 10 and in the late phase (d 100) after transplantation to assess for characteristics in the intrahepatic leukocyte (IHL) population in the phase of tolerance. METHODS: Lewis rats served as recipients of Dark Agouti rat livers. Temporary immunosuppression with either cyclosporine (CsA) monotherapy (3 mg/kg/d) or triple therapy that consisted of a subtherapeutic CsA dosage (0.25 mg/kg/d) and monoclonal antibodies directed against the interleukin-2 receptor (IL-2R, CD25) and the intercellular adhesion molecule-1 (ICAM-1, CD54) was administered from postoperative d 0 to d 13. Cell migration and cell activation within liver grafts was assessed by standard histology and flow cytometry. IHL apoptosis was detected by terminal deoxynucleotidyl transferase-mediated dUTP-digoxigenin nick end labeling (TUNEL). RESULTS: Both CsA monotherapy and triple therapy prolonged liver allograft survival to more than 100 d and led to the induction of donor-specific tolerance. Untreated recipients rejected their allografts within 14 d. In both groups, donor-specific IHLs initially dropped to 18% to 25% on d 10, but they rebounded to as much as 40% on d 100 as a common characteristic of both groups. Within this population, donor-specific T cells were dominant. In both groups, increased numbers of activated (IL-2R+) CD8+ T lymphocytes were present on d 100. No accumulation of apoptotic IHL was observed on d 100. Their proportion was unchanged in the triple therapy group and slightly decreased in the CsA group compared to the syngeneic controls. CONCLUSIONS: The present study reveals that tolerant liver allografts are repopulated by donor-specific T lymphocytes. This phenomenon is independent of the type of applied immunosuppression. The persistence of activated CD8+ T cells in the phase of proven donor-specific tolerance on d 100 indicates that liver tolerance is associated with the state of a permanent intragraft immune activation. It seems that the coexistence of donor cells with infiltrating recipient cells within liver grafts, termed intrahepatic cell chimerism, is characteristic for tolerated liver allografts.     

Engraftment following T-cell-depleted bone marrow transplantation. II. Stability of mixed chimerism in semiallogeneic recipients after total-body irradiation

Chronic, stable mixed chimerism of both lymphocytes and erythrocytes was observed in semiallogeneic murine recipients of T-cell-depleted bone marrow transplants that had been conditioned with supralethal total-body irradiation (1100 cGy). Mixed chimerism was extensive, with a wide range of donor engraftment persisting for at least one year after transplant. In both erythrocyte and lymphocyte lineages, decreasing donor engraftment correlated with decreasing marrow dose; however, complete red cell engraftment was more easily achieved than complete lymphocyte engraftment. There were no late graft failures, even among animals exhibiting a substantial host component of hematopoiesis. The extent of mixed hematopoietic chimerism therefore appears to be much greater than had been expected in recipients of T-cell-depleted bone marrow transplants.     

嵌合体在同种心脏移植免疫耐受中的作用

目的探讨嵌合体在同种心脏移植免疫耐受中的作用.方法采用大鼠腹部心脏移植模型,将30只Lewis大鼠随机分成正常对照组(Ⅰ组)、排斥反应组(Ⅱ组)、免疫耐受组(Ⅲ组),每组10只.观察移植心存活时间,供心病理学改变,供、受者间的混合淋巴细胞反应(MLR)和脾、胸腺嵌合体.结果Ⅲ组供心平均存活时间(85.28±7.48天)较Ⅱ组(7.33±1.03天)显著长(P＜0.01);Ⅱ组供心见大量炎性细胞浸润,Ⅲ组供心仅见少量炎性细胞浸润;Ⅲ组供、受者间MLR较Ⅰ组显著低(P＜0.01);Ⅲ组受者的脾、胸腺形成了稳定的供者细胞嵌合体.结论移植免疫耐受的受者形成了稳定的中枢和外周嵌合体,嵌合体的形成对移植耐受起重要的作用.     

Comparative analysis of immunological reconstitution induced by vascularized bone marrow versus bone marrow cell transplantation

Abstract  We have reported previously that vascularized bone marrow transplantation (VBMT) in an orthotopic hind limb graft brings about complete repopulation of bone marrow cavities in lethally irradiated syngeneic recipients within 10 days. Intravenous infusion of an equivalent volume of bone marrow cell suspension was evidently less effective. The purpose of this study was to investigate the reconstitution of immunocompetent compartments of lethally irradiated syngeneic rats after VBMT. Lewis rat hind limbs were transplanted orthotopically into irradiated recipients. Ten days after irradiation and bone marrow transplantation, bone marrow, mesenteric lymph nodes, and sera from rats were harvested. Mesenteric lymph node lymphocytes were analyzed. The responsiveness fomesenteric lymph node lymphocytes (MLNL) to mitogens and cell proliferation in the presence of sera and bone marrow cell (BMC) culture supernatants were measured. Our studies have shown that vascularized bone marrow transplantation brings about rapid replenishment of lymphoid organs of lethally irradiated syngeneic recipients. The re-populating subsets are fully responsive to mitogens. Sera from reconstituting rats had no effect on the proliferation of mature lymphocytes. Intravenous infusion of a number of BMC in suspension equivalent to that grafted in hind limb transplant was less efficient in reconstitution of lymphoid tissue.     

Allogeneic hematopoietic stem-cell transplantation, mixed chimerism, and tolerance in living related donor renal allograft recipients

OBJECTIVE: We designed a prospective, randomized, and controlled clinical trial to evaluate the efficacy and safety of achieving a mixed chimerism-associated tolerance protocol for recipients of living related donor (LRD) renal allografts. PATIENTS AND METHODS: Sixty-six consecutive patients were divided into two equal groups of 33 patients with end-stage renal disease. They were enrolled for transplantation after negative lymphocytotoxicity cross-matching (LCM). Both groups (treated [Tn] and control [Cn]) showed similar clinical and laboratory parameters and donor HLA match profiles. The Tn group underwent thymic transplantation of donor renal tissue, two donor-specific transfusions, low-intensity conditioning, and high-dose hematopoietic stem-cell transplantation (HSCT) before renal transplantation. The conditioning regimen included low-dose, target-specific irradiation (to abdominal and inguinal lymph nodes, bone marrow [BM] from thoracolumbar vertebrae and part of the pelvis on alternate days, 100 rad x 4), anti-T-cell antibodies (1.5 mg/kg body weight [BW]), cyclophosphamide (10 mg/kg BW x 2 consecutive days), and cyclosporine (CyA; >3 mg/kg BW/d). Unfractionated HSCT procured from the donor marrow was administered into the BM, portal and peripheral circulations, within 24 hours of achieving CD 4+/CD 8+ T-cell count less than 10% of normal. This infusion was supplemented with a dose of peripherally mobilized stem cells (mean total dose of 20 x 10(8) cells/kg recipient BW) administered peripherally. Renal transplantation was performed after negative LCM. Donor-specific cytotoxic antibodies were eliminated with intravenous immunoglobulins and plasmapheresis before renal transplantation. Mixed chimerism was evaluated before and after transplantation at monthly intervals in patients with donors of opposite gender by the FISH technique. Both groups received CyA and prednisolone for immunosuppression; Cn subjects also received mycophenolate mofetil/azathioprine. Rejection was treated with standard treatment. Immunosuppression was withdrawn 6 months after renal transplantation for patients with consistently positive chimerism. Clinical tolerance was defined as stable allograft function for more than 100 days without immunosuppression and confirmed by allograft biopsy. RESULTS: Over a mean follow-up of 210 days, all Tn patients showed stable allograft function with mean serum creatinines (SCr) of 1.20 mg/dL, no rejection/CMV infections/graft or patient loss. A low-level donor-specific cytotoxic antibody was observed in all Tn patients. The CyA toxicity was noted in 10 (30.3%) patients. Persistent mixed hematopoietic chimerism was seen in all 21 patients irrespective of donor-recipient HLA matching (mean 0.5% before and 1 +/- 0.3% after transplantation). All four patients on drug withdrawal have shown donor-specific tolerance at a mean follow-up of 129.8 days. Other Tn patients are in the process of being weaned off immunosuppression. Mean SCr of controls was 1.45 mg/dL over a mean follow-up of 216 days. Acute rejection was observed in 17 (51.5%) patients; no CMV infection/patient loss was noted and one (3.03%) graft was lost in controls. No patient was lost in controls. No graft-versus-host disease was observed in Tn patients. CONCLUSION: We have achieved mixed hematopoietic chimerism-associated tolerance with high-dose HSCT, intrathymic donor renal tissue transplantation, and minimal conditioning without any adverse effects.     

Mixed chimerism of cardiomyocytes and vessels after allogeneic bone marrow and stem-cell transplantation in comparison with cardiac allografts

Controversy persists about mixed chimerism (mCh) occurring in the hearts of patients after orthotopic cardiac transplantation in comparison with allogeneic bone marrow (BM) and peripheral blood stem-cell (PBSC) transplants. Cadaver hearts were examined after sex-mismatched transplantation by immunophenotyping combined with dual color fluorescence in situ hybridization (X and Y chromosome-specific probes). A striking disparity in the extent of mCh depending on the different transplantation procedures was recognizable. After allografting with PBSCs, 1.7% chimeric cardiomyocytes were detectable contrasting 5.4% of donor cells after full BM transplantation. In cardiac transplants, host-type endothelial cells (16.2%) and myocytes (14.3%) of the vessel walls were more often encountered than after BM and PBSC allografting. A sprouting of vascular structures into the donor heart after orthotopic cardiac transplantation has to be assumed, as does a pivotal role of the mesenchymal stem cells of the BM in the development of mCh.     

UV-B modulation of haplo-identical bone marrow cells in the prevention of GVHD and induction of specific transplantation tolerance to intestinal allografts

Ultraviolet B (UV-B) irradiation of BM cells (BMC) prior to transplantation into lethally gamma irradiated allogeneic rats prevents graft-versus-host disease (GVHD), induces stable allogeneic chimerism and specific tolerance to donor-type allografts. This study evaluated the role of UV-B modulation of bone marrow transplant (BMT) in the prevention of GVHD in the parent (P)-to-F1 hybrid rat combination of Lewis-to-Lewis × Brown Norway F1 (LBNF1) and of subsequent tolerance to small bowel transplantation (SBT). Lethally gamma irradiated (10.5 Gy) LBNF1 recipients of unmodified Lewis BMT (admixture of 10⁸ BMC and 5 × 10⁶ spleen cells) developed lethal GVHD and died within 55 days. Similarly, groups of lethally irradiated LBNF1 recipients of Lewis BMT treated with 100–300 J/m² UV-B developed GVHD and died within 47 days. All F1 hybrid recipients of Lewis BMT treated with 700 or 900 J/m² of UV-B permanently accepted their BM grafts, gained weight, showed no clinical evidence of GVHD and survived for > 300 days. These stable chimeras expressed 94–98% donor T-cells in their lymph nodes and became tolerant to BM donor-type (Lewis) SBT when grafted 180 days after BMT. In contrast, similarly prepared F1 recipients rejected BN SBT, thus demonstrating donor specificity. The chimeric rats were specifically unresponsive to donor (Lewis) Ag in MLR while they maintained normal alloreactivity to BN stimulators. These results suggest that UV-B irradiation of BMT offers a promising approach to overcoming the limitations of T-cell depletion of BMT and may offer a useful approach for recipient conditioning for induction of transplantation tolerance.     

一种大鼠带血管的骨髓移植模型

目的 构建一种简单可靠的带血管的骨髓移植模型.方法 将供体带血管蒂的股骨通过显微血管吻合的方式移植到受体腹股沟区,20只Lewis近交系大鼠随机分3组:同基因移植组Lewis→Lewis;排斥组Lewis→BN;免疫抑制组Lewis→BN,术后给予环孢素A.通过大体和病理学检查观察各组移植物存活情况,流式细胞计数监测外周血嵌合水平.结果 术后30 d,同基因移植组股骨存活良好,骨髓苏木素-伊红(HE)染色与正常骨髓无异,而排斥组第7天即发生显著的排斥反应,骨髓细胞明显减少、坏死,外周血嵌合水平几乎为0;和同系移植组一样,免疫抑制组术后30 d移植物存活良好,术后1、2、3、4周外周血中可检测到供体特异性嵌合,嵌合水平分别为(4.7±2.0)％、(2.2±1.2)％、(1.8±0.9)％、(1.5±0.3)％.结论 带血管蒂的股骨移植是一种简便可靠的骨髓移植模型,可作为一种新的骨髓移植方法诱导免疫耐受.     

Variable relationship between chimerism and tolerance after hematopoietic cell transplantation without myelosuppressive conditioning

BACKGROUND: We have previously described a mixed chimerism protocol that avoids myelosuppressive conditioning and permits hematopoietic cell transplantation across MHC barriers without the need for whole body irradiation in miniature swine. Here, we report our current experience including animals conditioned without thymic irradiation, and we attempt to define the relationship between long-term chimerism and stable tolerance in these animals. METHODS: Recipient swine received in vivo T-cell depletion, with or without thymic irradiation on day -2. Cyclosporine was administered for 30 to 60 days beginning on day -1. A total of 1 to 2 x 10(10) /kg cytokine-mobilized donor hematopoietic cells were infused during 3 days. Chimerism was determined by flow cytometry. In vitro tolerance assays and donor-matched kidney transplantation were performed after cessation of cyclosporine. RESULTS: Most recipients maintained stable chimerism (26 of 35) and were specifically tolerant to donor-matched cells in vitro regardless of whether they received thymic irradiation. Donor-matched kidney transplantations performed in chimeric animals without in vitro antidonor immune responses were accepted without immunosuppression. Some animals developed in vitro evidence of antidonor MHC responsiveness despite the persistence of donor cells in the peripheral blood. Donor-matched kidney transplantations performed in the face of these responses were rejected. CONCLUSIONS: These data indicate that this nonmyelosuppressive protocol can induce stable chimerism and robust tolerance even in animals conditioned without thymic irradiation. However, the data also demonstrate that macrochimerism does not always correlate with tolerance. Lack of in vitro antidonor immune responses in chimeric animals is an important predictor of renal allograft acceptance in this model.