Cyclosporine facilitates chimeric and inhibits nonchimeric tolerance after posttransplant total lymphoid irradiation

BACKGROUND: Previous studies showed that Lewis rats given posttransplant total lymphoid irradiation, antithymocyte globulin, and a single infusion of ACI peripheral blood or bone marrow cells develop tolerance to ACI heart allografts. METHODS: To determine the effects of cyclosporine on these tolerance induction protocols, groups of Lewis hosts, given either ACI blood or marrow infusions, were given a 60-day course of daily cyclosporine immediately after the cell infusion. RESULTS: Cyclosporine treatment was associated with uniform graft rejection in the groups given an ACI blood transfusion, and was associated with uniform graft acceptance in the groups given an ACI bone marrow infusion. Studies of donor-type T and B cell chimerism in the host blood showed that cyclosporine facilitated chimerism in the hosts given ACI bone marrow cells, and stable chimerism over a 300-day observation period was predicted by detectable chimerism by day 30. None of the hosts given ACI blood cells developed chimerism. CONCLUSION: Cyclosporine facilitated long-term graft acceptance in a tolerization protocol that induced mixed chimerism, but prevented long-term graft acceptance in a tolerization protocol that did not induce chimerism.     

Liver allografts are toleragenic in rats conditioned with posttransplant total lymphoid irradiation

BACKGROUND: Posttransplant total lymphoid irradiation (TLI) treatment has been applied to tolerance induction protocols in heart and kidney transplantation models. METHODS: We examined the efficacy and mechanism of posttransplant TLI treatment in the induction and maintenance of tolerance in a rat orthotopic liver transplantation model. RESULTS: Posttransplant TLI prolonged ACI (RT1(a)) liver allograft survival in Lewis (RT1(b)) hosts, with 50% long-term engraftment without immunosuppression and without evidence of chronic rejection. Injection of donor-type liver mononuclear cells (LMCs) facilitated the prolongation of graft survival, with more than 70% of grafts in LMC recipients surviving more than 100 days without chronic rejection. Recipients with long-term liver allograft survival accepted ACI but not PVG skin grafts. In TLI-conditioned recipients with accepted grafts, apoptosis occurred predominantly in graft-infiltrating leukocytes. In contrast, there were few apoptotic leukocytes in rejecting grafts. Recipients with long-term graft acceptance (>100 days of survival) demonstrated evidence of immune deviation; mixed lymphocyte reaction to ACI stimulator cells was vigorous, but secretion of interferon-gamma and interleukin-2 was reduced. In tolerant recipients, the number of Foxp3(+) CD25(+) CD4(+) regulatory T cells was increased in the liver allograft as well as in the peripheral blood. CONCLUSION: We conclude that posttransplant TLI induces tolerance to liver allografts via a mechanism involving apoptotic cell-deletion and immunoregulation.     

Mixed chimerism and immunosuppressive drug withdrawal after HLA-mismatched kidney and hematopoietic progenitor transplantation

BACKGROUND: Rodents and dogs conditioned with total-lymphoid irradiation (TLI), with or without antithymocyte globulin (ATG), have been shown to develop mixed chimerism and immune tolerance without graft-versus-host disease (GVHD) after the infusion of major histocompatability complex (MHC)-mismatched donor bone marrow cells given alone or in combination with an organ allograft. METHODS: Four human leukocyte antigen (HLA)-mismatched recipients of living donor kidney transplants were conditioned with TLI and ATG posttransplantation and infused with cyropreserved donor granulocyte colony-stimulating factor (G-CSF) "mobilized" hematopoietic progenitor (CD34+) cells (3-5x10(6) cells/kg) thereafter. Maintenance prednisone and cyclosporine dosages were tapered, and recipients were monitored for chimerism, GVHD, graft function, T-cell subsets in the blood, and antidonor reactivity in the mixed leukocyte reaction (MLR). RESULTS: Three of the four patients achieved multilineage macrochimerism, with up to 16% of donor-type cells among blood mononuclear cells without evidence of GVHD. Prolonged depletion of CD4+ T cells was observed in all four patients. Rejection episodes were not observed in the three macrochimeric recipients, and immunosuppressive drugs were withdrawn in the first patient by 12 months. Prednisone was withdrawn from a second patient at 9 months, and cyclosporine was tapered thereafter. CONCLUSIONS: Multilineage macrochimerism can be achieved without GVHD in HLA-mismatched recipients of combined kidney and hematopoietic progenitor transplants. Conditioning of the host with posttransplant TLI and ATG was nonmyeloablative and was not associated with severe infections. Recipients continue to be studied for the development of immune tolerance.     

Tolerance and Withdrawal of Immunosuppressive Drugs in Patients Given Kidney and Hematopoietic Cell Transplants

Sixteen patients conditioned with total lymphoid irradiation (TLI) and antithymocyte globulin (ATG) were given kidney transplants and an injection of CD34+ hematopoietic progenitor cells and T cells from HLA‐matched donors in a tolerance induction protocol. Blood cell monitoring included changes in chimerism, balance of T‐cell subsets and responses to donor alloantigens. Fifteen patients developed multilineage chimerism without graft‐versus‐host disease (GVHD), and eight with chimerism for at least 6 months were withdrawn from antirejection medications for 1–3 years (mean, 28 months) without subsequent rejection episodes. Four chimeric patients have just completed or are in the midst of drug withdrawal, and four patients were not withdrawn due to return of underlying disease or rejection episodes. Blood cells from all patients showed early high ratios of CD4+CD25+ regulatory T cells and NKT cells versus conventional naive CD4+ T cells, and those off drugs showed specific unresponsiveness to donor alloantigens. In conclusion, TLI and ATG promoted the development of persistent chimerism and tolerance in a cohort of patients given kidney transplants and hematopoietic donor cell infusions. All 16 patients had excellent graft function at the last observation point with or without maintenance drugs.     

Association of natural killer cell depletion with induction of mixed chimerism and allograft tolerance in non-human primates

BACKGROUND: Nonmyeloablative T cell depletion followed by donor bone marrow infusion has proved to be an effective approach to induction of mixed chimerism and tolerance of organ allografts in non-human primates. To help define the mechanisms involved we have compared T cell depletion with ATG versus anti-CD2 monoclonal antibody with respect to establishment of mixed chimerism and induction of tolerance. METHOD: Both nonmyeloablative regimens included low dose total body irradiation (1.5 Gy x 2), thymic irradiation (7 Gy), splenectomy and kidney plus donor bone marrow transplantation, followed by a 4-week posttransplant course of cyclosporine. In addition, the ATG group (13 recipients) received antithymocyte globulin, although the LOCD2b group (10 recipients) were treated with an anti-CD2 monoclonal antibody (LOCD2b). RESULTS: In the ATG group, 11 of 13 monkeys developed multilineage chimerism and 9 survived for more than 100 days without kidney allograft rejection. In contrast, 0/10 monkeys in the LOCD2b group developed chimerism, 5 died of infection and 5 suffered progressive rejection; only 1 recipient survived beyond 100 days. Sequential monitoring of peripheral blood mononuclear cells revealed greater T cell (CD3+) depletion in the LOCD2b-treated animals compared to those receiving ATG. However, NK cells (CD16+CD8+) were significantly more depleted in the ATG group and NK function remained abrogated longer after ATG than LOCD2b treatment (3 weeks vs. <5 days). CONCLUSION: Despite excellent T cell depletion by LoCD2b, ATG was more effective in inducing chimerism and tolerance. This difference correlated with anti-NK activity of the two reagents. These data suggest that NK cells may also resist engraftment of allogeneic bone marrow cells in this model.     

Facial tissue allograft transplantation

A hemifacial allograft transplant model was used to investigate the rationale for development of functional tolerance across an MHC barrier. Thirty hemiface transplantations were performed in five groups of six Lewis (RT1(1)) rat recipients each. Isografts were performed in group 1. Transplants were obtained from semiallogenic LBN(RT1(1+n)) in group 2 and from fully allogenic ACI(RT1(a)) in group 3 donors, which served as allograft rejection controls. Group 4 grafts using LBN donors and group 5 using ACI donors in addition received CsA monotherapy (16 mg/kg/d for 1 week) and maintained at 2 mg/kg/d. Signs of graft rejection were sought daily. Isograft controls survived indefinitely. All nontreated allografts were rejected within 5 to 8 days posttransplant. Eighty-three percent of face-transplant recipients from LBN donors and 67% from ACI donors did not show any signs of rejection up to 270 days and 200 days, respectively. Flow cytometry at day 63 in LBN recipients showed the presence of donor-specific chimerism for MHC class I RT1(n) antigens, namely 3.39% CD4/RT1(n); 1.01% CD8/RT1(n) T-lymphocytes; and 3.54% CD45RA/RT1(n) B-lymphocytes. In ACI recipients the chimerism test revealed 10.55% CD4/RT1(a) and 4.59% of CD8/RT1(a) T-lymphocytes. MLR assay at day 160 posttransplant revealed suppressed responses against LBN donor antigens in group 4, but moderate reactivity to ACI donor antigens in group 5. Functional tolerance toward hemifacial allograft transplants induced across MHC barrier using a CsA monotherapy protocol was associated with the presence of donor-specific chimerism in T- and B-cell subpopulations.     

Tolerance induced by bone marrow chimerism prevents transplant vascular sclerosis in a rat model of small bowel transplant chronic rejection

BACKGROUND: The major impediment to success in solid organ transplantation is chronic rejection (CR). The characteristic lesion of CR is transplant vascular sclerosis (TVS). Although the mechanism of TVS is thought to have an immunologic basis, in humans immunosuppression does not prevent or reverse it. One possible therapy to prevent TVS is induction of donor-specific tolerance. Bone marrow chimerism has been successful in inducing tolerance in acute and chronic rejection heart and kidney transplant models. The highly immunogenic small bowel (SB) allograft provides a rigorous test of the efficacy of this tolerance regimen. We examined whether induction of tolerance by bone marrow chimerism could prevent TVS in a model of Fisher 344 (F344) to Lewis (LEW) rat SB transplantation. METHODS: Bone marrow chimeras (BMC) were created by transplantation of T-cell-depleted F344 bone marrow into irradiated LEW rats. Chimerism was assessed by flow cytometric method. F344 SB, heterotopically transplanted into the chimeras, was clinically and histologically assessed for CR. F344 SB grafts, transplanted into cyclosporine-A-treated LEW recipients, served as control grafts for CR. RESULTS: Cyclosporine-A-treated LEW rats chronically rejected F344 SB grafts. By contrast, the BMC group demonstrated tolerance and had long-term SB graft survival (>120 days) without TVS. The BMC demonstrated immunocompetence by prompt rejection of third party ACI (RT1av1) SB allografts. CONCLUSIONS: Bone marrow chimerism prevents chronic graft failure secondary to TVS in a model of chronic SB rejection. TVS fails to develop when tolerance is established, suggesting that the mechanisms involved in TVS are, in part, immunologically mediated.     

Tolerance in a concordant nonhuman primate model

BACKGROUND: We have previously demonstrated that induction of mixed lymphohematopoietic chimerism resulted in donor specific renal allograft tolerance without the need for chronic immunosuppression in nonhuman primates. Here we have tested whether tolerance can be similarly induced for baboon to cynomolgus renal xenografts. METHODS: After preconditioning with anti-thymocyte globulin (ATG), nonlethal total body irradiation, and thymic irradiation, cynomolgus monkeys underwent splenectomy, native nephrectomies, and baboon marrow and renal transplants. Postoperative cyclosporine was given for 28 days. RESULTS: In Group 1 (n=2, survival= 13, 14 days), both animals developed anti-donor immunoglobulin G, had biopsy findings consistent with humoral rejection, and showed rapidly progressive xenograft failure. In Group 2 (n=5, survival=1, 16, 33, 112, 190 days), 15-deoxyspergualine was added to the regimen (Day 0-13). In one long-term survivor, donor specific hyporesponsiveness was first observed (mixed lymphocyte culture [(MLR]) on Day 48. MLR reactivity returned on Day 64 together with the development of anti-donor antibody and subsequent xenograft failure on Day 112. Donor specific T-cell hyporesponsiveness was detected in the other long-term survivor for the first 133 days, after which a donor-specific skin xenograft was placed, (survival 24 days). Following the skin graft rejection, a rise in the MLR, development of anti-donor antibody and progressive rejection of the renal xenograft were observed. CONCLUSIONS: Antibody-mediated rejection seems to constitute the major difference between concordant xenografts and allografts. Addition of 15-deoxyspergualine for 2 weeks posttransplant extended concordant primate xenograft survival to 6 months without chronic immunosuppression. In contrast to the allogeneic model, renal transplant acceptance in this xenogeneic system was interrupted by placement of a donor-specific skin graft.     

Long-term survival of xenogeneic heart grafts achieved by costimulatory blockade and transient mixed chimerism

BACKGROUND: Xenotransplantation holds great promise in clinical medicine, but is limited by the vigorous rejection response elicited against solid organs transplanted across species barriers. In this study, we investigated the role of anti-CD40L monoclonal antibody (mAb) in inducing xenogeneic mixed chimerism and donor-specific heart transplantation tolerance. METHODS: One day before heart transplantation, mice were injected intraperitoneally with anti-mouse CD8/NK1.1/Thy1.2 mAbs. On day 0, the mice received 3 Gy total body irradiation (TBI), an intravenous injection of unseparated bone marrow (BM) harvested from F344 rats, and an intraperitoneal injection of hamster antimouse CD40L mAb, MR1. Heart grafts from F344 rats were heterotopically transplanted into the abdomen of B6 mouse recipients. Using flow cytometric analysis of peripheral white blood cells, we assessed donor hematopoiesis at various times after bone marrow transplantation (BMT). RESULTS: Chimerism subsided gradually and disappeared completely 18 weeks after BMT. The cardiac graft survived permanently, even after the mixed chimerism disappeared. To determine if the mice acquired donor-specific tolerance, second rat heart grafts were transplanted 120 days after the first heart transplantation. The second transplanted hearts were also accepted over 60 days. Histological analysis revealed no remarkable vasculopathy in the coronary vessels at any stage. CONCLUSIONS: These findings clearly show that costimulatory blockade plays an important role in inducing xenochimerism, and that transient mixed chimerism can induce permanent acceptance of rat to mouse cardiac xenografts. Transplantation of xenogeneic bone marrow cells under costimulatory blockade at the time of heart transplantation may induce transplantation tolerance.     

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.     

Indirect allorecognition in acquired thymic tolerance: induction of donor-specific tolerance to rat cardiac allografts by allopeptide-pulsed host dendritic cells

BACKGROUND: Presentation of peptides either by recipient or donor MHC molecules displayed on the surface of antigen-presenting cells is an essential element in the induction of T cell responses to transplant antigens. The finding that intrathymic (IT) injection of an immunodominant peptide induces acquired thymic tolerance suggests an indirect pathway of allorecognition in the thymus. To address this theory, we studied the effects of IT injection of host bone marrow (BM)-derived dendritic cells (DC)-pulsed with the immunodominant Wistar Furth (WF) MHC class I (RT1.Au) peptide 5 (93-109) on cardiac allograft survival in the WF-to-ACI rat combination. METHODS: DC were propagated from cultures of ACI (recipient) bone marrow (BM) maintained in a medium supplemented with granulocyte-macrophage colony-stimulating factor and IL-4. The BM-derived DC after 8 days of culture were pulsed in vitro with a single WF MHC class I peptide (Residue 93-109) with the dominant epitope, washed, and injected into the thymus of ACI rats. The ACI recipients received donor-type (WF) or 3rd party (Lewis) cardiac allografts 7 days after IT immunization with peptide-pulsed DC. RESULTS: BM-derived DC cultured in granulocyte-macrophage colony-stimulating factor and interleukin-4 for 8 days have a strong allostimulatory ability and present peptide 5 to naive syngeneic T cells in mixed lymphocyte reaction. IT inoculation of 300 microg RT1.Au peptide 5 combined with transient antilymphocyte serum immunosuppressive therapy induced donor-specific tolerance to cardiac allografts. Extension of this finding to peptide-pulsed self DC showed that IT injection of peptide 5-pulsed host DC consistently led to permanent acceptance (>150 days) of donor-type (WF) cardiac allografts, whereas third-party (Lewis) grafts were acutely rejected. The long-term unresponsive recipients challenged with second-set grafts accepted permanently (>100 days) donor-type(WF) grafts while rejecting third-party (Lewis) grafts without the rejection of the primary WF grafts. CONCLUSION: This novel finding that allopeptide-pulsed host DC induces tolerance to cardiac allografts suggests that the induction of acquired tolerance is dependent on the indirect allorecognition pathway. The results further suggest that genetically engineered DC expressing donor MHC class I or II molecules or a peptide analogue might have therapeutic potential in the induction of transplant tolerance and in the treatment of autoimmune diseases.     

Kidney allograft survival in dogs treated with total lymphoid irradiation.

Total lymphoid irradiation (TLI) is immunosuppressive and, in rodent, can induce a state where transplantation of allogeneic bone marrow results in chimerism and permanent acceptance of organ allografts from the donor strain. We attempted to apply this treatment to a large animal model. Twelve splenectomized dogs were treated with TLI (150 rads per fraction, total dose 1950-3000 rads) before bilateral nephrectomy and renal allotransplantation. Eight dogs received bone marrow from the kidney donor. In 13 untreated control dogs renal allografts functioned (serum creatinine level less than 2.0 mg/dl) for a mean +/- (SE) of 4.7 +/- 0.3 days. In the four TLI treated dogs who did not receive bone marrow the renal allografts functioned for 15-76 days (two dogs died with functioning grafts). In the eight TLI treated dogs who received donor bone marrow, two died immediately after transplantation, two rejected at 3 and 13 days, one died at 13 days with a functioning graft, and two have had the grafts function for longer than 500 days. Chimerism was not detected in the one dog tested. The response of peripheral blood lymphocytes to stimulation with phytohemagglutinin and in mixed lymphocyte culture was suppressed for at least on month after TLI. The results confirm the immunosuppressive effect of TLI. The absence of kidney rejection in two recipients of donor bone marrow show the potential of this approach to induce long-term immunologic unresponsiveness as to an organ allograft, but the outcome is unpredictable and further experiments are needed to define the optimal conditions for administration of TLI and bone marrow to the recipients.     

Tolerance induction permits the development of graft-versus-host disease: donor-mediated attack following small bowel transplantation in mixed chimeras.

The induction of tolerance to organ allografts would eliminate acute and chronic rejection as well as the need for nonspecific immunosuppression. We have shown that tolerance induced through the creation of mixed allogeneic bone marrow chimeras allows for the long-term engraftment of cardiac and small bowel allografts across strong multiple major histocompatibility barriers. The possibility that tolerance might render the host susceptible to graft-versus-host disease (GVHD) has not been investigated in this or other models of tolerance. To test this possibility chimeras were created by transplantation of T-cell depleted ACI and Lewis bone marrow into lethally irradiated Lewis rats. Chimerism was determined post bone marrow transplant (BMTx) by flow cytometry of lymphocytes from reconstituted animals. ACI/Lew chimeras (ALC), Lewis/ACI F1 (LACF1), and Lewis (LEW) rats all received heterotopic ACI vascularized small bowel grafts. A second group of chimeras received small bowel grafts from ACI rats pretreated with low dose irradiation to eliminate T-cells from the graft. LEW-->LEW small bowel isografts were also performed. Animals were examined for evidence of GVHD by clinical signs and histologic examination of biopsied tissues. GVHD was quantified using the popliteal lymph node enlargement assay. All LACF1 rats developed severe lethal GVHD following ACI small bowel transplant. Bone marrow chimeras, ALC (n = 6), developed fatal GVHD in a similar fashion after receiving a small bowel transplant. LEW-->LEW isografts and chimeras receiving bowel from irradiated ACI rats survived long term without GVHD while ACI-->LEW allogeneic transplants all underwent acute rejection. GVHD or its absence was confirmed histologically. Popliteal lymph node enlargement indices reflected the presence of GVHD in the chimeras (1.87) and LACF1 (5.4) receiving allografts, but not in isografts or chimeras receiving irradiated allogeneic transplants. Analysis of cytokines in the tongues of rats undergoing GVHD showed elaboration of Th1 type proinflammatory cytokines which was not seen in isografted rats or rats receiving preirradiated small bowel. These results demonstrate that tolerance induction through mixed chimerism results in susceptibility to small bowel induced GVHD. Preirradiating the donor bowel prior to SBTx can prevent GVHD.     

Composite tissue allotransplantation in chimeric hosts part II. A clinically relevant protocol to induce tolerance in a rat model

BACKGROUND: We and others have shown that mixed allogeneic chimerism induces donor-specific tolerance to composite tissue allografts across major histocompatibility complex barriers without the need for immunosuppression. However, a delay period between bone marrow transplantation and limb allotransplantation is required, making such protocols impractical for clinical application. This study eliminates this delay period in a rat hind limb allotransplantation model by performing mixed allogeneic chimerism induction and transplantation "simultaneously." METHODS: Group 1 included controls in which na?ve Wistar Furth (WF) hosts received ACI hind limbs. Group 2 included (ACI-->WF) chimeras that received limbs from third-party donors (Fisher), and group 3 included chimeras that received irradiated (1,050 cGy) ACI limbs. In group 4, WF hosts conditioned with 950 cGy received irradiated (1,050 cGy) ACI limbs followed by infusion of 100 x 10(6) ACI T-cell-depleted bone marrow cells and immunotherapy (tacrolimus and mycophenolate mofetil) for 28 days. Group 5 animals received the same treatment as group 4 animals without immunotherapy. RESULTS: The rats in groups 1 and 2 rejected their limbs within 10 days. Only one rat in group 4 survived to the end of the study. Groups 3 and 5 demonstrated long-term limb survival without rejection or graft-versus-host disease. High levels of donor chimerism (>80%) were achieved and maintained throughout the study. Mixed lymphocyte reaction assays in both groups revealed donor-specific hyporesponsiveness with vigorous third-party reactivity. CONCLUSIONS: This study demonstrated that infusion of donor bone marrow cells into conditioned hosts immediately after limb transplantation results in stable mixed chimerism, robust tolerance, and reliable limb allograft survival.     

Allogeneic versus semiallogeneic F1 bone marrow transplantation into sublethally irradiated MHC-disparate hosts. Effects on mixed lymphoid chimerism, skin graft tolerance, host survival, and alloreactivity

In models of tolerance associated with mixed lymphoid chimerism, depletion of Thy 1+ cells from the allogeneic donor inoculum may decrease the level of chimerism achieved and the capacity of donor cells to induce tolerance. To determine whether the apparent role of Thy 1+ cells in the facilitation of bone marrow engraftment and induction of skin graft tolerance is related to alloaggression, the capacity of fully allogeneic C57BL/6J, H-2b BM cells to establish mixed lymphoid chimerism and skin graft tolerance in sublethally irradiated (2.5 Gy x 3) BALB/c, H-2d hosts was compared with that of semi-allogeneic BALB/c x C57BL/6J F1 H-2d/b BM cells which genetically lack the potential for graft-versus-host reactivity against parental recipients. The levels of mixed chimerism observed with allogeneic and semi-allogeneic F1 BM cells were nearly identical: 21.0 +/- 9.7% of spleen cells in H-2b BM-injected and 18.6 +/- 8.8% of spleen cells in H-2d/b BM-injected H-2d hosts were of donor allotype. There was no difference in the fraction of hosts rendered tolerant to C57BL/6J, H-2b skin grafts by H-2b vs. H-2d/b BM at either excess (94% vs. 92% tolerant) or threshold (37% vs. 40% tolerant) numbers of donor cells. Spleen cells from both types of mixed chimeras failed to respond to donor antigens in MLR. Both H-2b and H-2d/b BM-injected H-2d hosts rejected third party C3H/HeJ, H-2k skin grafts and responded to third party stimulators in MLR. Although these nonspecific allo-immune responses were not as strong as the responses of normal animals, they were suppressed to an equivalent degree in both types of chimeras. Graft-versus-host disease, if present in irradiated H-2b BM-injected hosts, did not significantly affect survival compared with survival of irradiated H-2d/b BM-injected animals. These results suggest that the tolerizing capacity of allogeneic BM does not depend upon GVHD and that allogeneic and semi-allogeneic BM establish mixed lymphoid chimerism and induce skin graft tolerance by similar mechanisms across a complete MHC disparity in sublethally irradiated adult hosts.     

Hybrid resistance to parental bone marrow grafts in nonlethally irradiated mice

Resistance to parental bone marrow (BM) grafts in F1 hybrid recipients is due to natural killer (NK) cell–mediated rejection triggered through “missing self” recognition. “Hybrid resistance” has usually been investigated in lethally irradiated F1 recipients in conjunction with pharmacological activation of NK cells. Here, we investigated BM‐directed NK‐cell alloreactivity in settings of reduced conditioning. Nonlethally irradiated (1‐3 Gy) or nonirradiated F1 (C57BL6 × BALB/c) recipient mice received titrated doses (5‐20 x 10⁶) of unseparated parental BALB/c BM without pharmacological NK cell activation. BM successfully engrafted in all mice and multilineage donor chimerism persisted long‐term (24 weeks), even in the absence of irradiation. Chimerism was associated with the rearrangement of the NK‐cell receptor repertoire suggestive of reduced reactivity to BALB/c. Chimerism levels were lower after transplantation with parental BALB/c than with syngeneic F1 BM, indicating partial NK‐mediated rejection of parental BM. Activation of NK cells with polyinosinic–polycytidylic acid sodium salt poly(I:C), reduced parental chimerism in nonirradiated BM recipients but did not prevent hematopoietic stem cell engraftment. In contrast, equal numbers of parental lymph node cells were completely rejected. Hence, hybrid resistance leads to incomplete rejection of parental BM under reduced conditioning settings.     

Mixed allogeneic chimerism as a reliable model for composite tissue allograft tolerance induction across major and minor histocompatibility barriers

BACKGROUND: Although prolonged composite tissue allograft (CTA) survival is achievable in animals using immunosuppressive drugs, long-term immunosuppression of CTAs in the clinical setting may be unacceptable for most patients. The purpose of this study was to develop a model for reliable CTA tolerance induction in the adult rat across a major MHC mismatch without the need for long-term immunosuppression. METHODS: Mixed allogeneic chimeras were prepared by using rat strains with strong MHC incompatibility [WF (RT1Au), ACI (RT1Aa)] WF + ACI-->WF, n=23. The bone marrow (BM) of recipient animals was pretreated with low-dose irradiation (500-700 cGy), followed by reconstitution with a mixture of T cell-depleted syngeneic (WF) and allogeneic (ACI) cells. Additionally, the recipient animals received a single dose of anti-lymphocyte serum (10 mg) 5 days before bone marrow transplantation (BMT) and tacrolimus (1 mg/kg/day) from the day before BMT to 10 days post-BMT. Hindlimb transplants were performed 12 months after BMT. Five animals received a limb allograft irradiated (1000 cGy) just before transplantation. Rat chimeras were characterized (percentage of donor cells present within the bloodstream) by flow cytometry at 3 and 12 months after BM reconstitution and after hindlimb transplantation. RESULTS: Peripheral blood lymphocyte chimerism (WF/ACI) remained stable >12 months after BM reconstitution in 18/23 animals. Multi-lineage chimerism of both lymphoid and myeloid lineages was present, suggesting that engraftment of the pluripotent rat stem cell had occurred. In animals with donor chimerism >60% (n=18) no sign of limb rejection was present for the duration of the study. All animals with chimerism <20% (n=5) developed moderate signs of rejection clinically and histologically. Gross motor and sensory reinnervation (weight bearing, toe spread) developed at >60 days in 14/21 rats. Postoperative flow cytometry studies demonstrated stable chimerism in all animals studied (n=10). Five out of five animals with irradiated limb transplants showed no sign of GVHD at >100 days. CONCLUSIONS: Stable mixed allogeneic chimerism can be achieved in a rat hindlimb model of composite tissue allotransplantation. Hindlimb allografts to mixed allogeneic chimeras exhibit prolonged, rejection-free survival. Partial functional return should be expected. The BM transplanted as part of the hindlimb allograft plays a role in the etiology of GVHD. Manipulating that BM before transplantation may influence the incidence of GVHD. This represents the first reliable rat hindlimb model demonstrating rejection-free CTA survival in an adult animal across a major MHC mismatch without the long-term need for immunosuppressive agents.     

Myeloma Responses and Tolerance Following Combined Kidney and Nonmyeloablative Marrow Transplantation: In Vivo and In Vitro Analyses

Six patients with renal failure due to multiple myeloma (MM) received simultaneous kidney and bone marrow transplantation (BMT) from HLA-identical sibling donors following nonmyeloablative conditioning, including cyclophosphamide (CP), peritransplant antithymocyte globulin and thymic irradiation. Cyclosporine (CyA) was given for approximately 2 months posttransplant, followed by donor leukocyte infusions. All six patients accepted their kidney grafts long-term. Three patients lost detectable chimerism but accepted their kidney grafts off immunosuppression for 1.3 to >7 years. One such patient had strong antidonor cytotoxic T lymphocyte (CTL) responses in association with marrow rejection. Two patients achieved full donor chimerism, but resumed immunosuppression to treat graft-versus-host disease. Only one patient experienced rejection following CyA withdrawal. He responded to immunosuppression, which was later successfully withdrawn. The rejection episode was associated with antidonor Th reactivity. Patients showed CTL unresponsiveness to cultured donor renal tubular epithelial cells. Initially recovering T cells were memory cells and were enriched for CD4+CD25+ cells. Three patients are in sustained complete remissions of MM, despite loss of chimerism in two. Combined kidney/BMT with nonmyeloablative conditioning can achieve renal allograft tolerance and excellent myeloma responses, even in the presence of donor marrow rejection and antidonor alloresponses in vitro.     

Treatment of rat heart allograft recipients with postoperative total lymphoid irradiation (TLI)

Pretransplant total lymphoid irradiation (TLI) plus donor bone marrow can result in donor-specific tolerance, but graft-versus-host disease is not consistently avoided. TLI may have greater applicability as an adjuct to generalized immunosuppressive treatment. In a clinical trial, preoperative TLI posed problems for patients with a high percentage of cytotoxic antibodies who sometimes had a long wait for a transplant after completion of TLI. It would be logistically advantageous if TLI could be given in the perioperative period. We investigated the feasibility of this approach in an ACI (RT-1^a) to Lewis (RT-1^l) rat heart allograft model. Untreated recipients rejected the grafts at a mean of 6.1 days. TLI 400 or 600 rads administered as one dose 1 day preoperative did not prolong graft survival (rejected a mean of 5.6 and 6.3 days). A dose fraction schedule previously found to be effective when given preoperatively, 200 rads × 5 consecutive days, did not prolong graft survival when administered beginning the first day postoperatively (mean rejection at 6.3 days). Higher dose fractions were effective: 300 rads × 3, 300 rads × 5, 400 rads × 3, and 400 rads × 4 gave mean rejection times of 11.8, 16.8, 11.5, and 13.6 days, respectively. Combining 600 rads preoperative with 200 rads × 5 days postoperative did not prolong graft survival. Six hundred rads preoperative plus 300 rads × 3 days postoperative prolonged graft survival, but was no more effective than 300 rads × 3 days postoperative alone. The most effective schedule for postoperative TLI was 300 rads × 3, 0 rads × 2, and 200 rads × 5 days; this treatment delayed rejection to 25 days. In conclusion, one dose preoperative TLI has no effect on heart allograft survival, but postoperative TLI in dose fractions of 300 rads is effective in delaying rejection. Postoperative TLI may be an alternative treatment for patients who must wait for suitable crossmatch negative cadaver kidneys.     

Dog leukocyte antigen-haploidentical stem cell allografts after anti-CD44 therapy and nonmyeloablative conditioning in a preclinical canine model

BACKGROUND: We previously described a reduced-intensity hematopoietic cell transplantation (HCT) regimen in dog leukocyte antigen (DLA)-haploidentical littermate recipients consisting of 450 cGy total body irradiation (TBI) and anti-CD44 monoclonal antibody (mAb) S5 before and mycophenolate mofetil (MMF)/cyclosporine (CSP) after HCT. METHODS: We tested a nonmyeloablative regimen of mAb S5 and 200 cGy TBI with postgrafting MMF/CSP in 44 DLA-haploidentical recipients using eight different regimens. Ten dogs also received escalating doses of donor lymphocyte infusion (DLI) alone or with pentostatin to convert to complete donor chimerism. RESULTS: All dogs achieved initial engraftment between one to two weeks after HCT with peripheral blood mononuclear cell (PBMC) donor chimerism ranging from 2% to 98% (median 37%) on day +35. Twenty-five (57%) dogs rejected their donor grafts at a median of seven (range; 1-19) weeks after HCT. Low levels of PBMC donor chimerism at three weeks predicted graft rejection. DLI neither facilitated conversion to full donor chimerism after HCT nor prevented rejection. Higher total nucleated cells, CD4+, CD8+, and CD14+ cell subset numbers in the PBMC graft were associated with stable full donor engraftment. Dogs given higher cell subset doses of infused PBMC achieved longer duration of mixed chimerism. CONCLUSIONS: Nonmyeloablative conditioning with 200 cGy TBI and anti-CD44 mAb was sufficient for initial uniform engraftment across DLA haplotype-mismatched barriers. However, sustained donor engraftment was seen in only 43% of recipients. Graft composition and donor-dominant chimerism early after HCT may be the most important factors for sustained donor engraftment.