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Background: Despite broad and intense conventional immunosuppression, long-term survival after lung transplantation lags behind that for other solid organ transplants, primarily because of allograft rejection. Therefore, new strategies to promote lung allograft acceptance are urgently needed. The purpose of the present study was to induce allograft tolerance with a protocol compatible with deceased donor organ utilization., Methods: Using the major histocompatibility complex-mismatched mouse orthotopic lung transplant model, we investigated a conditioning regimen consisting of pretransplant T cell depletion, low-dose total body irradiation and posttransplant (donor) bone marrow, and splenocyte infusion followed by posttransplantation cyclophosphamide., Results: Our results show that C57BL/6 recipients of BALB/c lung allografts undergoing this complete short-duration nonmyeloablative conditioning regimen had durable lung allograft acceptance. Mice that lacked 1 or more components of this regimen exhibited significant graft loss. Mechanistically, animals with lung allograft acceptance had established higher levels of donor chimerism, lymphocyte responses which were attenuated to donor antigens but maintained to third-party antigens, and clonal deletion of donor-reactive host Vβ T cells. Frequencies of Foxp3 T regulatory cells were comparable in both surviving and rejected allografts implying that their perturbation was not a dominant cell-regulatory mechanism. Donor chimerism was indispensable for sustained tolerance, as evidenced by acute rejection of allografts in established chimeric recipients of posttransplantation cyclophosphamide after a chimerism-ablating secondary recipient lymphocyte infusion., Conclusions: Together, these data provide proof-of-concept for establishing lung allograft tolerance with tandem donor bone marrow transplantation using a short-duration nonmyeloablative conditioning regimen and posttransplant cyclophosphamide.

Background: Cyclophosphamide (CP)-induced tolerance is a mixed chimerism-based tolerance induction protocol. Recently, we reported that invariant natural killer T (iNKT) cells were essential for the tolerance induction in this system. In this study, we evaluated the roles of the cytokines produced by iNKT cells., Methods: DBA/2 (H-2d) mice and BALB/c (H-2d) wild-type (WT) or iNKT knock out (KO) mice were used as donors and recipients. WT recipients received three doses (days -7, -4, -1 or 35, 38, 41) or a single dose (day -1 or 0) of alpha-galactosylceramide (GC) in conjunction with our conditioning regimen that consisted of 10(8) donor spleen cells on day 0 and 200 mg/kg CP on day 2. To investigate the iNKT cell function, iNKT KO recipients were reconstituted with cytokine (interferon-gamma, interleukin [IL]-4, or IL-10) KO iNKT cells and received donor spleen cells and CP., Results: Mixed chimerism was observed in WT recipients, but was reduced in iNKT KO recipients. However, mixed chimerism was absent in WT recipients given GC on days -7, -4, -1, but not in WT recipients given GC on day 35, 38, 41. Donor skin grafts were chronically rejected when mixed chimerism was diminished. Skin grafts were accepted in iNKT KO recipients reconstituted with iNKT cells from interferon-gamma, IL-4, or IL-10 KO mice and receiving our conditioning regimen., Conclusions: Invariant NKT cells were required in the initial phase of the induction of chimerism. Our results indicated that known major cytokines produced by iNKT cells were dispensable for the regulatory function of iNKT cells.

Background: Bone marrow (BM) chimerism has been shown to have a beneficial effect on allograft survival. We recently found that production of donor T-cells was highly correlated with induction of tolerance in minimally conditioned chimeras. In the present studies, we demonstrate that nonmyeloablative conditioning and BM cell infusion modulate innate and adaptive host immune responses., Methods: Chimeras were generated by bone marrow transplantation (B10.BR to B10). Recipients were preconditioned with T-cell depleting antibodies and total body irradiation with or without cyclophosphamide. Donor-specific tolerance was tested by skin grafting., Results: Transfer of tolerant splenocytes to immunocompetent secondary recipients did not transfer tolerance, nor did infusion of tolerant CD4+/CD25+ T-cells into chimeras without donor T-cell production, demonstrating that linked suppression is an unlikely mechanism in tolerance induction in the context of BM cell infusion. The addition of a single dose of cyclophosphamide to the conditioning enhanced engraftment and tolerance. This was associated with production of donor T-cells and effective clonal deletion, and a significant reduction in activated recipient plasmacytoid dendritic cells (pDC) and natural killer (NK) cells. Chimeras without donor T-cell production that eventually lost their chimerism did not generate an antidonor humoral response, whereas unconditioned controls infused with similar numbers of BM cells did, indicating that infusion of donor BM cells into conditioned recipients induced immune deviation for adaptive B-cell immunity, preventing sensitization to major histocompatibility complex (MHC) alloantigens., Conclusions: These results demonstrate that recipient T-cells, pDC, and NK cells contribute to the host barrier for establishing chimerism, implicate deletional tolerance as the mechanism for total body irradiation-based nonmyeloablative conditioning for BM transplantation, and show a beneficial effect of BM cells in preventing sensitization to MHC alloantigens.

Background: Animal allogeneic bone marrow transplantation (BMT) models with nonmyeloablative conditioning regimens have so far required irradiation or antibodies in addition to immunosuppressive drugs for engraftment. Moreover, although it is known that the balance between donor T-cell number and the dose of immunosuppressive drugs would be critical for engraftment, it has not been experimentally clarified in a nonmyeloablative regimen., Methods: We used C57BL/6 mice as donors and DBA/2 mice as recipients with a nonmyeloablative regimen including fludarabine (Flu) and cyclophosphamide (CPA) without irradiation or antibodies. To determine the adequate doses, we injected recipients with various doses of Flu and CPA, and 2x10 bone marrow cells (BMC) and 5x10 splenocytes (SC). Furthermore, using T-cell-depleted BMC and enriched T cells, we investigated the balance between donor T-cell number and the dose of Flu., Results: Doses of Flu at 150 mg/kg/dayx6 and CPA at 150 mg/kg/dayx2 were most appropriate for engraftment with low mortality. All mice appropriately pretreated and transplanted with both BMC and SC exhibited complete donor chimeras. Donor cell engraftment was not enhanced by any increase of BMC transplanted, and dose escalation of donor T cells but not BMC led to the reduction of Flu dose required for engraftment of donor cells., Conclusions: We have established a murine nonmyeloablative BMT model in a fully MHC-mismatched combination for donor cell engraftment with complete donor chimerism. Simultaneously, we have quantitatively demonstrated that the balance between donor T-cell number and the dose of immunosuppressive drugs is critical for stable engraftment.

Background: Hepatic veno-occlusive disease is a major limiting factor of high-dose chemotherapy in children. The cells lining the hepatic vascular endothelium express blood group A and/or B antigens according to the patient's blood group. We designed a study evaluating the impact of platelet concentrates containing ABO-incompatible plasma transfused to young children with a high risk of hepatic veno-occlusive disease., Methods: In all, 186 consecutive children (median age: 4 years, range: 0.75-17 years), treated with high-dose chemotherapy containing busulfan followed by hematopoietic stem cell transplantation for neuroblastoma (n=112) or brain tumor (n=74) between 1988 and 1998, were investigated. The main endpoint was the occurrence of hepatic veno-occlusive disease. Multivariate analysis was performed using a Cox's regression model with transfusion of platelet concentrates containing ABO-incompatible plasma as a time-dependent covariate., Results: We found that 73 out of 186 (39%) children developed hepatic veno-occlusive disease after transplantation. Multivariate analysis demonstrated that two factors significantly increased the risk of hepatic veno-occlusive disease occurrence: transfusion of platelet concentrates containing ABO-incompatible plasma (P=0.003) and use of melphalan in the conditioning regimen (P=0.006). Conversely, the number of platelet concentrates transfusions per week, child's age, weight, sex, and use of cyclophosphamide in the conditioning regimen had no effect., Conclusions: Transfusion of platelet concentrates containing ABO-incompatible plasma increases the risk of hepatic veno-occlusive disease in young children treated with a busulfan-containing regimen. Binding of A and/or B antigens expressed on the surface of hepatic endothelial cells may promote this complication. Transfusion of platelet concentrates containing ABO-incompatible plasma should be avoided in these children.

Background: Whether mixed chimeras induced by nonmyeloablative conditioning are tolerant to challenge with donor allogeneic islet grafts is unknown. Here we investigate whether our nonmyeloablative, costimulation blockade-free and sirolimus (SRL)-based protocol could facilitate mixed chimerism via bone marrow transplantation (BMT) and induce islet allograft tolerance., Methods: After low dose (1-3 Gy) total body irradiation (TBI, day -1), with or without prior lymphocyte depletion, C57BL/6 mice were transfused with 40 x 10(6) BALB/c bone marrow cells (day 0) and received SRL (3 mg/kg/day) for 4 weeks. Chimerism was monitored by flow cytometry and the recipients were rendered diabetic chemically and challenged with donor islets., Results: Mixed chimerism was achieved in mice treated with TBI 3 Gy/SRL but it declined over time in 60% (9/15) of them. Long-term stable chimerism was established in 100% of recipients over 50 weeks with either antilymphocyte serum (ALS, 9/9), anti-CD4 (4/4), or anti-CD4 plus anti-CD8 (5/5) prior to BMT. TBI conditioning could be reduced to 1 Gy, with 90% (9/10) maintaining chimerism in the long-term. When TBI was substituted with cyclophosphamide (CTX) or busulfan (BUS), all mice remained chimeric in the long-term. The chimeras showed no proliferative response to donor antigen and accepted both first and second donor-specific islet grafts indefinitely while rejecting third-party grafts., Conclusions: This data provides the first evidence that stable fully allogeneic chimeras induced with BMT after nonmyeloablative conditioning with SRL and lymphocyte-depleting antibodies exhibit robust donor-specific tolerance to islet grafts.

Background: We investigated an approach to separating graft-versus-lymphoma (GVL) effects from graft-versus-host disease (GVHD) in mice receiving a nonmyeloablative conditioning regimen allowing establishment of mixed hematopoietic chimerism., Methods: We evaluated the ability of donor lymphocyte infusions (DLI) to mediate GVL effects without GVHD in mixed chimeras prepared with cyclophosphamide, anti-T-cell antibodies, and thymic irradiation. To examine the fate of GVH-reactive donor CD8+ T cells, we used the 2C T-cell receptor (TCR) transgenic mouse strain, which carries an Ld-specific transgenic TCR on the B6 background., Results: Administration of DLI on day 35 post-BMT led to conversion from mixed to full donor chimerism and mediated a powerful GVL effect with complete protection (100% survival) against mortality induced by a host-type lymphoma (EL4) administered 7 days later (100% mortality in non-DLI controls; P<0.001). No GVHD occurred in DLI recipients. Rechallenging the surviving DLI recipients, which had converted to full chimerism, with the same tumor dose 17 weeks later led to rapid tumor mortality. Long-term DLI recipients had anti-host proliferative responses, but not CTL responses in vitro. When given as DLI together with wild-type spleen cells, marked expansion of GVH-reactive 2C CD8+ T cells was observed on day 10, followed by a marked decline in their numbers by week 10 post-DLI., Conclusions: Nonmyeloablative induction of mixed chimerism followed by administration of DLI can mediate powerful GVL effects. The late loss of DLI-mediated GVL effects may reflect the eventual loss of donor-derived GVH-reactive CTL, which occurs in association with conversion to full donor chimerism.

Background: T-cell-depleted bone marrow transplantation (TDBMT) can prevent graft-versus-host disease (GvHD). However, depleting T cells from allogeneic bone marrow often results in failure of bone marrow engraftment under irradiation conditioning. It is not know whether donor T cells are essential for bone marrow engraftment and whether blocking the CD40/CD154 pathway promotes allogeneic TDBM engraftment under nonmyeloablative and irradiation-free fludarabine phosphate and cyclophosphamide conditioning therapy., Methods: Using fully major histocompatibility complex (MHC)-matched mouse models, we investigated whether donor T cells are essential for bone marrow engraftment under fludarabine phosphate and cyclophosphamide conditioning therapy. We also determined whether the barrier of allogeneic TDBM could be overcome by blocking the CD40/CD154 pathway. Donor chimerism was detected by flow cytometric analysis. Donor-specific tolerance through establishing mixed chimerism was tested in vivo by skin transplantation and in vitro by mixed leukocyte reaction and enzyme-linked immunospot (ELISPOT) assay., Results: Compared with unmodified bone marrow, TDBM resulted in poor engraftment when fully MHC-mismatched donors were used. However, anti-CD154 monoclonal antibody (mAb) treatment significantly enhanced donor TDBM engraftment. TDBM engraftment was also seen in CD154 knockout mice. A stable and high level of multilinage donor chimerism was achieved. Recovery of host CD3 T cells was suppressed, and recovery of donor CD3 T cells was promoted, after TDBMT and anti-CD154 mAb treatment. Donor chimerism was established by TDBMT induced donor-specific tolerance in vivo and in vitro., Conclusion: Donor T cells facilitate bone marrow engraftment under nonmyeloablative and irradiation-free conditioning therapy, and the blocking the CD40/CD154 pathway can replace donor T cells to promote TDBM engraftment.

Stem-cell transplantation from human leukocyte antigen (HLA)-haploidentical family members carries a high risk of rejection and graft-versus-host disease (GVHD) if donor and recipient differ by more than one HLA antigen. The authors have developed treatment protocols from studies in dog leukocyte antigen-haploidentical dogs that prevent rejection and modify GVHD to the extent that patients with aggressive hematologic neoplasia can be treated with success. Principal improvements have been achieved in the use of cyclophosphamide and total-body irradiation for conditioning and T-cell depletion for prevention of GVHD. More recently, the combination of marrow and CD6-depleted mobilized donor blood cells (MDBC) has been introduced for HLA-haploidentical transplantation on the basis that CD6-depleted MDBC contain immunoregulatory cells besides stem cells and natural killer cells. Clinical results are reported on 36 patients with high-risk hematologic neoplasia. The results encourage the use of HLA-haploidentical stem-cell transplantation at an earlier stage of the disease. This method could also be of use for tolerance induction in organ transplantation.

Background: Human type 1 diabetes is associated with defects in the hematopoietic stem cells. Simultaneous donor islet and bone marrow transplantation may be an ideal therapeutic approach for inducing tolerance to islet allogeneic antigens and restoring self-tolerance to islet autoimmune antigens., Methods: Using a nonobese diabetic (NOD) mouse model of human type 1 diabetes, we investigated whether tolerance to MHC-matched allogeneic islet grafts from male nonobese diabetes-resistant (NOR) donors can be induced in female NOD recipients by simultaneous islet and bone marrow transplantation under fludarabine phosphate-based nonmyeloablative and irradiation-free conditioning therapy. Donor-specific chimerism in the peripheral blood of tolerant mice (n=7) was measured by semiquantitative polymerase chain reaction for a male-specific marker (SRY)., Results: Donor-specific tolerance to NOR islet grafts was induced in all diabetic NOD mice after simultaneous islet and bone marrow transplantation and treated with fludarabine phosphate, cyclophosphamide, anti-mouse lymphocyte serum, and rapamycin. At 100 days and 200 days after transplantation, the average percentage of male NOR marker in DNA derived from the peripheral blood of NOD recipients that had long-term islet graft survival was over 10%., Conclusion: Our data suggest that this approach may induce donor-specific tolerance in clinical islet transplantation and living-related donor solid organ transplantation.

Background: Reactive oxygen species (ROS) and glutathione (GSH) depletion contribute to organ injury after bone marrow transplantation (BMT). Keratinocyte growth factor (KGF) ameliorates graft-versus-host disease (GVHD)-associated organ injury in murine BMT models., Methods: B10.BR mice received total body irradiation (TBI; day -1) +/- cyclophosphamide (Cy; 120 mg/kg/day i.p., days -3 and -2), then were transplanted on day 0 with C57BL/6 bone marrow + spleen cells as a source of GVHD-causing T cells. KGF (5 mg/kg/day subcutaneously [s.c.]) or saline was given on days -6, -5, and -4. Lung and liver GSH and oxidized GSH disulfide (GSSG) levels were measured on days 0 and 5 and glutathione redox potential (Eh) calculated. Organ malondialdehyde (MDA) was determined on day 5 as an index of ROS-mediated lipid peroxidation., Results: In lung, TBI+BMT oxidized GSH Eh and increased MDA. Cy further oxidized lung GSH Eh. In liver, neither BMT regimen altered GSH redox status or MDA. KGF prevented the decrease in lung GSH after TBI+Cy and decreased lung MDA after both TBI and TBI+Cy. KGF increased liver GSH levels and GSH Eh after TBI and GSH Eh after TBI+Cy., Conclusions: In murine allogeneic BMT, TBI oxidizes the lung GSH redox pool and Cy exacerbates this response by 5 days post-BMT. In contrast, liver GSH redox status is maintained under these experimental conditions. KGF treatment attenuates the Cy-induced decrease in lung GSH, decreases post-BMT lung lipid peroxidation, and improves liver GSH redox indices. KGF may have a therapeutic role to prevent or attenuate GSH depletion and ROS-mediated organ injury in BMT.

Background: The induction of tolerance to pig antigens in primates may facilitate the development of successful clinical xenotransplantation protocols. The infusion of mobilized porcine peripheral blood leukocytes (PBPCs, comprised of approximately 2% peripheral blood progenitor cells) into splenectomized preconditioned (whole body irradiation (WBI)-based) baboons, intended to induce mixed hematopoietic cell chimerism, however, results in a severe thrombotic microangiopathy (TM) that includes pronounced thrombocytopenia. Previous studies have indicated that the infused PBPCs initiate platelet aggregation, but that the various individual components of the conditioning regimen are not associated with the development of aggregation. We have now investigated the effects of cyclophosphamide (CPP) as an alternative to WBI on platelet aggregation., Methods: Splenectomized baboons (n=3) were treated with CPP. Blood samples were collected and platelet-rich plasma (PRP) was prepared. Using light transmission aggregometry, the extent of aggregation induced by platelet agonists (thrombin, adenosine diphosphate (ADP), collagen, ristocetin, and arachidonic acid) was determined in vitro. PRP was also prepared from untreated baboons and from baboons receiving CPP, PBPCs were added, and platelet aggregation was measured in the absence of exogenous platelet agonists., Results: CPP markedly inhibited platelet aggregation induced by all standard agonists. In vitro addition of PBPCs to PRP stimulated platelet aggregation in the absence of any agonists. Prior treatment of baboons with CPP, however, inhibited this effect by 55% to 65%. TM was not evident in baboons receiving a conditioning regimen that included CPP instead of WBI., Conclusions: Aggregation of baboon platelets and TM is directly induced by PBPCs. CPP has direct anti-aggregatory properties and may provide an alternative strategy to WBI in this pig-to-primate model intended to induce mixed hematopoietic cell chimerism.

Background: We previously established a cyclophosphamide (CP)-induced tolerance system in rodent skin graft models. In this study, we applied this system to rat liver transplantation., Methods: Lewis recipients were inoculated on day -2 with spleen and bone marrow cells (SC+BMC) from Dark Agouti (DA) donors, followed by 100 mg/kg CP on day 0. On day 25, DA livers were orthotopically grafted. We assessed the alloresponses to the donors of the long-term surviving recipients, using the second skin grafting and in vitro assay., Results: The recipients that had been treated with SC+BMC and CP survived for more than 165 days. None of control group that received SC+BMC alone (mean survival times [MST]=13.8 days), CP alone (MST=40.0), SC+BMC from third-party PVG rats and CP (MST=45.0), or no treatment (MST=13.8) survived over 50 days. The donor-specific tolerance was confirmed by second skin grafts onto recipients with permanent DA liver grafts, which accepted DA skins (MST>75) but not PVG (MST=8.3). However, the lymphocytes from the tolerant recipients showed alloresponse to DA in vitro. To investigate whether the T helper type 2 deviation contributed to this "split tolerance," we assessed the production of cytokines in mixed lymphocyte reaction. Interleukin 2 and interferon-gamma were detected but interleukin 4 was not., Conclusions: These data showed that this protocol induced split tolerance in rat liver transplantation and, furthermore, the mechanism of split tolerance was not due to T helper 2 deviation.

We elucidated the possible role of chimerism in skin and heart allograft tolerance using cyclophosphamide (CP)-induced tolerance. When C3H (H-2k; Thy1.2, Mls-1b) mice were i.v. primed with 1x10(8) spleen cells (SC) from H-2 matched AKR (H-2k; Thy1.1, Mls-1a) mice and then treated i.p. with 200 mg/kg of CP, the survivals of both AKR skin grafts and heart grafts (HG) were permanently prolonged in a tolerogen-specific fashion. After this treatment, a minimal degree of mixed chimerism, the clonal destruction of Mls-1a-reactive CD4+Vbeta6+ T cells in the periphery, and the clonal deletion of Vbeta6+ thymocytes were all observed. When AKR SC and 100 mg/kg CP were used for conditioning, the AKR HG were permanently accepted, but the survival of the AKR skin grafts was only mildly prolonged. The clonal destruction of CD4+Vbeta6+ T cells in the periphery and the intrathymic clonal deletion of Vbeta6+ thymocytes were induced in both the SC and the 100 mg/kg CP-treated C3H mice. A minimal degree of mixed chimerism was detectable at 4 and 12 weeks after AKR SC and 100 mg/kg CP treatment, and still did not disappear at 40 weeks. The degree of mixed chimerism induced with SC and 100 mg/kg CP was significantly lower than that with SC and 200 mg/kg CP during the observation. No posttransplant cardiac allograft vasculopathy (CAV) was observed to develop, while both the Th1 type (interferon-gamma) and Th2 type (interleukin-4 and -10) cytokine expressions decreased in the AKR HG of the tolerant C3H mice treated with both AKR SC plus 200 mg/kg CP, and AKR SC plus 100 mg/kg CP. A second set of skin grafts from donor AKR mice survived for more than 100 days in a tolerogen-specific fashion in all C3H mice treated with AKR SC and 200 mg/kg CP and also accepted the AKR HG for over 200 days, while 80% of the C3H mice treated with AKR SC and 100 mg/kg CP and accepted the AKR HG for more than 200 days. These results strongly suggested the following conclusions: 1) the degree of chimerism can strongly influence the induction of skin and heart allograft tolerance, 2) posttransplant CAV does not develop in the donor HG maintained by chimerism-based CP-induced tolerance, 3) the mRNA expression of both Th1 and Th2 type cytokine decreased in the donor HG maintained by chimerism-based CP-induced tolerance, and 4) the induction of skin allograft tolerance is more difficult than the prevention of posttransplant CAV.

Background: Bone marrow transplantation (BMT) and gene therapy are potent approaches to the recovery of bone marrow depression and induction of antitumor immunity after chemotherapy for the treatment of malignancies. In the present study, enhanced antitumor effect of BMT in combination with fibroblast-mediated interleukin (IL)-2 and IL-3 gene therapy was observed in tumor-bearing mice after chemotherapy., Methods: BALB/c mice were inoculated s.c. with J558L plasmacytoma cells and injected i.p. with cyclophosphamide 300 mg/kg 3 days later. 24 hours after chemotherapy syngeneic bone marrow cells in combination with NIH3T3 fibroblast cells engineered to produce IL-2 (NIH3T3-IL-2) and/or NIH3T3 cells engineered to produce IL-3 (NIH3T3-IL-3) were implanted into the tumor-bearing mice., Results: BMT in combination with implantation of either NIH3T3-IL-2 or NIH3T3-IL-3 cells exerted significant inhibition on the growth of J558L tumors and prolonged the survival period of the tumor-bearing mice as compared with the treatments with Hanks solution, BMT alone, or BMT plus implantation of NIH3T3 cells transduced with Neo gene. Synergistic antitumor effect was observed in mice after combined BMT and cytokine gene therapy. The cytotoxicities of natural killer cells, cytotoxic T lymphocytes, and macrophages in mice increased markedly after the combined treatment. Recovery of CFU-GM, CFU-MK and CFU-E formation in mice after combined therapy was accelerated obviously in mice after combined therapy., Conclusions: BMT in combination with fibroblast-mediated IL-2 and IL-3 gene therapy elicited augmented antitumor effects synergistically in tumor-bearing mice after chemotherapy mainly through induction of antitumor immune response and accelerated recovery of hematopoiesis.

Background: The supply of solid organs for transplantation will never meet the growing demand. Xenotransplantation is considered to be a potential solution for the critical shortage of allografts. However, xenograft rejection is currently not controlled by conventional immunosuppressive agents. Bone marrow chimerism induces donor-specific tolerance without the requirement for chronic immunosuppressive therapy. The aim of this study was to develop a nonlethal recipient-conditioning approach to achieve mixed bone marrow chimerism and donor-specific tolerance., Methods: C57BL/10SnJ mice were conditioned with total body irradiation followed by a single injection of cyclophosphamide on day +2. On day 0, mice were reconstituted with untreated bone marrow cells from Fischer 344 rats. Recipients were analyzed by flow cytometry for donor bone marrow engraftment and multilineage chimerism. Donor-specific tolerance was tested by skin grafting., Results: One hundred percent of recipients engrafted after irradiation with 600 cGy total body irradiation, transplantation with 80 x 10(6) Fischer 344 bone marrow cells, and injection with 50 mg/kg cyclophosphamide intraperitoneally. Donor chimerism was detectable in all engrafted animals for up to 11 months. This conditioning was nonlethal, because conditioned untransplanted animals survived indefinitely. Mixed xenogeneic chimeras were tolerant to donor-specific skin grafts but rejected third-party (Wistar Furth) grafts as rapidly as naive C57BL/10SnJ mice. In contrast, animals that received less efficacious conditioning regimens and did not exhibit detectable chimerism showed prolonged graft survival, but delayed graft rejection occurred in all animals within 10 weeks., Conclusion: The induction of bone marrow chimerism and donor-specific tolerance after nonlethal conditioning might be useful to prevent the vigorous cellular and humoral rejection response to xenografts.

The long-term success of organ transplantation is limited by complications resulting from consistent nonspecific immunosuppression. Induction of stable, donor-specific tolerance remains the main goal of transplantation immunology. In this article, a new, nonmyeloablative method is described for induction of transplantation tolerance to fully mismatched bone marrow cells (BMC), bone marrow stromal precursors, heart muscle, and skin allografts. The method is based on pretransplant conditioning with no postgraft immunosuppression, and consists of a short course (six daily fractions of 200 cGy) of total lymphoid irradiation (sTLI), followed by selective elimination of donor-specific alloreactive cells of the host escaping low-dose sTLI. Donor-specific alloreactive cells were activated by intravenous inoculation with a high dose of donor BMC (3 x 10(7) cells) 1 day after sTLI, and eliminated by a single intraperitoneal dose (200 mg/kg) of cyclophosphamide given 1 day after cell transfer. Infusion of a low number of T cell-depleted BMC (3 x 10(6) cells) after tolerogenic preconditioning converted recipients to stable mixed chimeras free of graft-versus-host disease. The same treatment provided long-lasting acceptance of heterotopically transplanted allografts of the heart muscle and of the stromal precursors to the hematopoietic microenvironment. This treatment also led to acceptance and life-long survival of full-thickness donor skin allografts. However, skin allografts survived only in mice that received donor T cell-depleted BMC after cyclophosphamide and had 20-50% donor cells in the blood. Our results suggest that after sTLI, additional selective clonal deletion of residual host cells induces a state of long-lasting specific tolerance to a wide variety of donor-derived tissues.

In normal rats, the xenobiotic K76 inhibited the C5 and probably the C2 and C3 steps of complement and effectively depressed classical complement pathway activity, alternative complement pathway activity, and the C3 complement component during and well beyond the drug's 3-hr half-life. It was tested alone and with intramuscular tacrolimus (TAC) and/or intragastric cyclophosphamide (CP) in rat recipients of heterotopic hearts from guinea pig (discordant) and hamster (concordant) donors. Single prevascularization doses of 100 and 200 mg/kg increased the median survival time of guinea pig hearts from 0.17 hr in untreated controls to 1.7 hr and 10.2 hr, respectively; with repeated injections of the 200-mg dose every 9-12 hr, graft survival time was increased to 18.1 hr. Pretreatment of guinea pig heart recipients for 10 days with TAC and CP, with or without perioperative splenectomy or infusion of donor bone marrow, further increased median graft survival time to 24 hr. Among the guinea pig recipients, the majority of treated animals died with a beating heart from respiratory failure that was ascribed to anaphylatoxins. Hamster heart survival also was increased with monotherapy using 200 mg/kg b.i.d. i.v. K76 (limited by protocol to 6 days), but only from 3 to 4 days. Survival was prolonged to 7 days with the addition of K76 of intragastric CP at 5 mg/kg per day begun 1 day before operation (to a limit of 9 days); it was prolonged to 4.5 days with the addition of intramuscular TAC at 2 mg/kg per day beginning on the day of transplantation and continued indefinitely. In contrast to the limited efficacy of the single drugs, or any two drugs in combination, the three drugs together (K76, CP, and TAC) in the same dose schedules increased median graft survival time to 61 days. Antihamster antibodies rapidly increased during the first 5 days after transplantation, and plateaued at an abnormal level in animals with long graft survival times without immediate humoral rejection. However, rejection could not be reliably prevented, and was present even in most of the xenografts recovered from most of the animals dying (usually from infection) with a beating heart. Thus, although effective complement inhibition with K76 was achieved in both guinea pig- and hamster-to-rat heart transplant models, the results suggest that effective interruption of the complement cascade will have a limited role, if any, in the induction of xenograft acceptance.

We have previously reported a method for inducing tolerance using cyclophosphamide (CP) in a murine model, in which 200 mg/kg of CP is administered intraperitoneally 2 days after intravenous priming with donor spleen cells. The CP-induced tolerance method, however, cannot induce long-lasting skin allograft survival in an MHC class I-disparate combination. In this study, we tried to explain this phenomenon based on a costimulatory theory. That is, allo-class I-reactive host CD8+ helper T cells may receive insufficient costimulatory signals from donor spleen cells and, therefore, they are resistant to subsequently administered CP. We demonstrated that activated donor B cells, which can deliver sufficient costimulatory signals, induce a more efficient proliferation of allo-class I-reactive host CD8+ helper T cells than naive B cells in vitro. In addition, we also demonstrated that our idea can also be applied to the CP-induced skin allograft tolerance in an MHC class I-disparate combination.

Treatment of adult mice with high doses of the immunosuppressive drug cyclophosphamide (CY) induces transient splenic natural suppressor (NS) cell activity mediated largely by cells bearing the MAC-1+ cell-surface marker. Here we show that culture supernatants from mixed lymphocyte reactions (MLR) suppressed by MAC-1+ NS cells exhibit decreased IL-2 and IL-4 activity in bioassays for these lymphokines. However, inhibition of MLR was maximal whether the regulatory cells were added at initiation of culture or 24 hr postinitiation, suggesting that inhibition of lymphokine synthesis is not likely to be the reason for diminished lymphocyte proliferation, since these particular lymphokine genes are known to be transcribed and expressed during the first 12 hr of culture. Furthermore, flow cytofluorometric analysis demonstrated that the presence of MAC-1+ NS cells did not alter the percentage of lymphokine-producing CD4+ T cells in MLR. IL-2 receptor (p55) expression was also normal in suppressed MLR. The addition of exogenous IL-2 and/or IL-4 to MLR failed to reverse the inhibitory effect of MAC-1+ NS cells on lymphocyte proliferation, indicating that these regulatory cells block the utilization of these lymphokines in MLR. The inhibitory effect of MAC-1+ NS cells on lymphocyte proliferation in MLR is dependent on interferon-gamma, since NS activity was dramatically decreased in the presence of neutralizing antibodies to interferon-gamma. MAC-1+ NS cell-induced suppression of MLR was also diminished in the presence of indomethacin, suggesting that prostaglandins play a role in this NS system.

The survival of skin grafts from B6.C-H-2bm1 (bm1; Kbm1,IAb,IE-,Db) mice was prolonged when C57BL/6 CrSlc (B6; H-2b) mice were inoculated intravenously with 9 x 10(7) spleen cells (SC) plus 3 x 10(7) bone marrow cells from bm1 mice 14 days prior to skin grafting. When B6 mice were inoculated i.v. with bm1 cells and treated intraperitoneally with 200 mg/kg cyclophosphamide (CP) 2 days later, the survival of bm1 skin grafts was not prolonged at all, suggesting antagonism between the veto cell-mediated enhancement by donor cells and CP-induced enhancement. In order to deplete the veto cells from the tolerogen, SC plus BMC were treated with anti-Thy1.2 mAb+C' or CP. The survival of the bm1 skin grafts was not prolonged at all in B6 mice inoculated with Thy1.2-treated bm1 or bm1 cells from CP-treated donors. When B6 mice were inoculated with Thy1.2-treated or CP-treated bm1 cells and followed by CP treatment, however, the survival of bm1 skin grafts was prolonged moderately. These results strongly suggested that the unresponsiveness induced with donor-derived veto cells prevents the tolerance induction to class I alloantigens (H-2Kbm1) by our protocol of CP-induced tolerance. Furthermore, in B6 mice injected with anti-Thy1.2 mAb on day -1 and bm1 cells on day 0, the survival of bm1 skin grafts was not prolonged at all. However, skin graft tolerance to bm1 antigens was induced when B6 mice were injected with anti-Thy1.2 mAb on day -1 and bm1 cells on day 0 followed by CP on day 2. These results may also be explained by the depletion of donor-derived veto cells in the recipient mice.

Based on previous studies showing that allogeneic islets transplanted into the thymus can induce donor-specific unresponsiveness, we investigated in the nonobese diabetic (NOD) mouse the effect of intrathymic islet isografts on preventing autoimmunity directed against pancreatic islet antigens. Islets prepared from newborn NOD pancreata were injected into one lobe of the thymus of 10- to 11-day-old female NOD mice (experimental group) with no immunosuppression. PBS alone was used for injection into age- and sex-matched litter mates (control group). Thirty of 32 (94%) experimental mice remained normoglycemic for over 30 weeks. Well-formed islets with no indication of insulitis were found in the thymus of these 30 mice, whereas no grafted islets were found in the 2 mice that became diabetic at 17 and 19 weeks, respectively (technical failures). In the control group, 10 of 32 (31%) mice became diabetic between 20 and 29 weeks. This diabetic incidence was, however, lower than that in our colony female mice. In the pancreas of experimental mice, 90.9% of islets were free of infiltrates, whereas only 13.1% of islets were intact in control mice. The spleens of 30-week-old experimental mice contained a slightly higher percentage of CD8+ T cells (P < 0.05) than those of control mice. Cyclophosphamide injections at 30 weeks induced diabetes in 4 of 9 experimental mice. The 2 lines of evidence, (1) marked reduction in insulitis of intrathymic islet-grafted mice and (2) induction of diabetes after treatment with cyclophosphamide, suggest that both thymic clonal deletion and peripheral tolerance may play a role in preventing diabetes.

Studies of the myeloablative regimens capable of permitting successful BMT across MHC barriers in miniature swine have been performed. To minimize graft-versus-host disease (GVHD), engraftment was studied in the F1-->P combination (i.e., MHC homozygous ["parental"] swine receiving bone marrow from one-haplotype matched MHC heterozygous ["F1"] donors). Animals given total body irradiation (TBI) up to 1100 cGy, 10 cGy/min, in a single dose failed to engraft. Increasing the dose rate led to unacceptable extramedullary toxicity without improving engraftment. Eleven different fractionated TBI regimens were tested in this F1-->parent model. At all of the dose rates tested, a total dose of less than 1000 cGy was insufficient for engraftment, and a total dose of 1400 cGy led to unacceptable toxicity. Between these extremes, a window was defined in which engraftment could be obtained without unacceptable extramedullary toxicity utilizing 2 equally divided fractions of TBI delivered 24 hr apart. The addition of 50 mg/kg cyclophosphamide i.v. to fractionated TBI (1150 cGy total dose [500 + 650]) also permitted engraftment, with decreased incidence of interstitial pneumonitis as compared to fractionated TBI (1300 cGy total dose [650 x 2]). Both of these regimens were also confirmed to permit engraftment between heterozygous donors and recipients sharing a single common haplotype ("F1-->F1"). The regimen of 1300 cGy (650 x 2) also permitted engraftment in completely MHC mismatched BMT, but with subsequent death from GVHD. These studies of the myeloablative regimens permitting engraftment across defined MHC barriers in miniature swine provide a basis for further studies of allogenic BMT and GVHD in this large animal preclinical model.

Antibody and complement have been shown to be of primary importance in the rejection of hamster heart xenografts by rats. Very high anti-hamster antibody titers were detected at the time of rejection of hamster hearts transplanted into untreated or T cell deficient rats. This study demonstrates a method of inhibiting this antibody production by pulse therapy with cyclophosphamide (CyP) and continuous cyclosporine treatment, resulting in a median survival of the hamster heart of greater than 100 days. Controls and CsA-treated rats reject the transplanted hamster heart in a median of 3 days. CyP as a sole therapy resulted in a median survival of 14 days. Prolonged CyP therapy when combined with CsA was associated with increased death among rat recipients due to infection. Antispecies antibody production was suppressed during CyP and CsA therapy and did not recur after cessation of CyP therapy. Cessation of CsA therapy at 60 and 100 days posttransplantation resulted in subsequent rejection of the xenografts (median survival after cessation of therapy of 11 and 19.5 days, respectively) and was associated with production of rat anti-hamster antibodies.

Transplantation tolerance across H-2D plus IE antigen barriers has been achieved when B10.Thy1.1 (Kb, IAb, IE-, Db; Thy1.1) mice were primed i.v. with 9 x 10(7) spleen cells plus 3 x 10(7) bone marrow cells from B10.A(5R) (Kb, IAb, IEb, Dd; Thy1.2) and treated i.p. with 200 mg/kg of cyclophosphamide (CP) two days later. The tolerant state was confirmed by prolonged acceptance of donor-type skin grafts, and in vitro unresponsiveness to donor antigens. From the early stage of tolerant state, V beta 11+ or V beta 5+ T cells expressing CD4 or CD8 accessory molecules were markedly decreased in the periphery of the tolerant mice. Moreover, neither CD4+CD8- nor CD4-CD8+ thymocytes bearing a high density of V beta 11 or V beta 5 were detected in the chimeric thymus. The intrathymic clonal deletion appeared to be maintained in some of the recipient mice even after the disappearance of detectable mixed chimerism in the late stage. These results suggest that the mechanisms of the CP-induced tolerance include the destruction of the IE (and probably H-2D) reactive T cells in the periphery followed by the intrathymic clonal deletion of T cells reactive against these antigens. These results directly show the strong correlation between transplantation tolerance to H-2 alloantigens and the disappearance of alloreactive T cells in both the periphery and thymus.

We reviewed the medical records of 97 patients undergoing T cell-depleted allogeneic bone marrow transplantation at our institution from 1984 to 1990 to determine the incidence of hepatic dysfunction, including venoocclusive disease of the liver following BMT. All patients received allogeneic marrow that had been purged with monoclonal antibody to the CD6 surface antigen (T12) and rabbit complement as the sole method of graft-versus-host disease prophylaxis. No additional immunosuppressive agents were routinely administered to these patients. Overall, 55% of patients in our series developed two-fold elevations in serum bilirubin, SGOT, or alkaline phosphatase within the first 30 days following BMT. A five-fold elevation in any liver function test was noted in only 19% of patients. Logistic regression analysis revealed that the presence of GVHD, female sex, and administration of amphotericin B all were independently associated with laboratory evidence of hepatic dysfunction. While LFT abnormalities were common in our series, they were generally mild, and the development of VOD was rare. Only three patients (3.1%) fulfilled clinical criteria sufficient to establish a diagnosis of VOD. Among the 86 patients whose ablative regimen consisted of cyclophosphamide (60 mg/kg x2) and total-body irradiation (1200-1400 cGy in 200 cGy fractions), only 1 patient (1.2%) developed VOD. Our experience suggests that patients undergoing allogeneic BMT are at low risk for VOD and other serious hepatic complications when they receive high-dose cyclophosphamide, fractionated TBI, and T cell-depleted marrow without hepatotoxic medications for GVHD prophylaxis.

BALB/cxDBA/2Wf F1 (CD2F1) mice were lethally irradiated and reconstituted with syngeneic bone marrow cells untreated or treated with 75 micrograms/ml of mafosfamide. One day after bone marrow transplantation some groups of mice were injected with syngeneic splenocytes, peripheral blood leukocytes, or thymocytes. Seven days after marrow grafting the anti-L1210 leukemia immunization of mice, consisting of four i.p. injections of 10(6) L1210-Maf cells (L1210 cells treated in vitro with mafosfamide for inhibition of their growth in vivo), was started. Strong resistance against leukemia could be obtained only in mice receiving splenocytes or peripheral blood leukocytes, not in mice injected with thymocytes or in those not receiving any cells. In vitro elimination of various subpopulations from among the splenocytes before their injection into the mice made it possible to deduce which are necessary for early induction of antitumor resistance after bone marrow transplantation in mice. These cells are: Thy 1.2-, Ig-, AsGM 1-, Mac 1+, 1-Ad+/-, are adherent and nonsusceptible to carrageenan toxicity.

Four skin graft models are created which vary in the rates of lymphatic and vascular reconstitution. Rat allografts (Brown Norway or (L times BN)F1 hybrid) transplanted to Lewis recipients by conventional (immediate lymphatic, delayed vascular), surgical anastomosis (immediate lymphatic, immediate vascular) or isolated anastomosis (delayed lymphatic, immediate vascular) techniques have similar mean survival times (7.8 days). Grafts placed on an isolated recipient pedicle (delayed lymphatic, delayed vascular) double the mean survival time to 15.6 days. Treatment with alloimmune serum, able to indefinitely prolong survival of similarly mismatched renal grafts, prolongs only isolated anastomosed grafts. In contrast, cyclophosphamide treatment prolongs survival of all groups. These results suggest, first, that both vascular and lymphatic routes of sensitization are equally effective and, second, that immunological enhancement requires either prompt vascular continuity or a persistent lack of lymphatic reconstitution.

The present experiment was designed to study the relationship between rat heart allograft survival and passenger leukocyte depletion in donor-pretreated animals. Untreated Lewis rats served as recipients of cardiac allografts from treated Fischer rats. Passenger leukocyte depletion was assayed with indium-111 oxine-labeled leukocytes (predominantly lymphocytes) which were infused into donor rats 6 hr before treatment with cyclophosphamide, antilymphocyte globulin (ALG), sublethal total body irradiation, or in vitro perfusion-preservation of the isolated beating heart. In vivo pretreatment of the donor with cyclophosphamide resulted in a significant prolongation of heart allograft survival but effected no reduction in graft-labeled lymphocytes. In vitro perfusion-preservation of the donor heart, for 1 to 2 hr, led to a 50 to 60% reduction in graft-labeled lymphocytes but failed to significantly prolong the survival of the heart allografts. Both ALG and sublethal total body irradiation donor pretreatments resulted in significant prolongation of heart allograft survival and a 20 to 25% labeled passenger lymphocyte depletion. This study demonstrates that there is no direct correlation between allograft survival and the degree of mobile passenger lymphocyte depletion, suggesting that the efficacy of leukocytotoxic donor pretreatment methods may depend in part on alternative mechanisms.

In a randomized study ww have been unable to demonstrate any beneficial effect of cadaver donor pretreatment with 5 g of methylprednisolone and 7 g of cyclophosphamide on graft function or survival in 22 recipients of pretreated kidneys compared with 30 recipients of nonpretreated kidneys.

F1 mice receiving sublethal whole-body x-irradiation (300 rads) or treatment with cyclophosphamide prior to the i.p. inoculation of parental spleen cells developed fatal graft-versus-host disease (GVHD). A greater survival rate was obtained when the inoculated parenteral spleen cells were obtained from BCGcw-preimmunized donors. The immunization of the F1 host with bacillus Calmette-Guerin cell walls (BCGcw) also increased host survival. The combined treatment of preimmunizing the host with BCGcw and of using spleen cells from BCGcw-immunized parental donors to initiate the GVHD resulted in producing the least severe GVHD and the greatest overall survival. The systemic transfer of x-irradiated spleen cells from BCGcw-immunized parental mice inhibited the fatal GVHD induced by the inoculation of normal parental spleen cells. These studies show that BCGcw immunization of the host or obtaining parental spleen cells from BCGcw-immunized animals resulted in improving the overall survival rate in graft-versus-host disease. BCGcw immunization induces suppressor cells and the decrease of graft-versus-host disease that was observed was most likely due to the induction of suppressor cells.

We have investigated the effects of a single injection of cyclophosphamide (CY) in F1 host mice. One day after CY treatment, F1 host mice show increased susceptibility to graft-versus-host (GVH)-reactive parental strain spleen cells when measured by the popliteal lymph node (PLN) enlargement assay. F1 host mice challenged with parental strain cells 7 days after CY treatment show decreased PLN enlargement as compared with CY-untreated F1 hosts receiving parental strain cells. The PLN reactivity of normal F1 spleen cells injected into F1 hosts pretreated with CY was also measured. F1 spleen cells elicited marked PLN enlargement when injected into F1 hosts that were treated with CY 1 day previously. By day 7 after CY treatment of F1 hosts, F1 cells no longer produced significant PLN enlargement as compared to untreated F1 hosts receiving F1 cells. We discuss these results in terms of the possibility that CY initiates changes in host antigenicity that can lead both to an increase in responsiveness of GVH-reactive cells and to susceptibility to attack by syngeneic cells.

B cell chimerism was analyzed in juvenile chickens given injections of major histocompatibility complex-compatible lymphoid cells. The allelic products of donor and host-derived B cells were monitored with antisera directed against M1 (IgM) and G1 (IgG) isotype-specific allotypes. Injection of peripheral blood lymphocytes and spleen cells suppressed host allotype levels whereas purified T lymphocytes were ineffective. Pretreatment of recipients with cyclophosphamide was more effective than irradiation in promoting both engraftment and host B cell suppression. Host allotype suppression endured for several months after cell transfer and was attributable to B cell deletion, as ascertained by the lack of cells which expressed surface M1. In partially suppressed chickens, host G1 was inhibited to a greater degree than M1 allotype. Host recovery was followed by donor B cell rejection when low numbers of donor cells and/or inadequate host conditioning was used. Selective M1 chimerism occurred when limiting numbers of spleen cells were transferred into cyclophosphamide-treated hosts, whereas selective G1 chimerism resulted after the transfer of large numbers of peripheral blood lymphocytes or spleen cells into unconditioned recipients. We consider that these findings can be attributed to a B cell surveillance mechanism which may be similar to that postulated previously to explain resistance to haemopoietic and tumour cells.

The maintenance of skin allotolerance induced by perimetamorphic application of MHC-disparate skin to isogeneic Xenopus is investigated. Removal of the perimetamorphically applied first-set graft after 4 weeks did not, in general, completely break allotolerance; however, many second-set semi-allogeneic grafts, applied up to 14 weeks after first-set removal, were no longer maintained in perfect condition. Skin allografts tolerated for up to 42 weeks continued to express donor histocompatibility antigens, as indicated by their survival times when transplanted back to the original donor or recipient strain. Treatment with human recombinant IL-2 (rIL-2), shown elsewhere to be an effective immunoregulatory lymphokine for Xenopus in vivo, failed to cause long-term-tolerated 1st-set allografts, or newly-applied 2nd-set grafts, to be rejected. In contrast, cyclophosphamide (CyP) treatment led to acute (less than 4 weeks) destruction of both 1st- and 2nd-set allografts; breaking of tolerance was regularly seen when donor and host differed by two MHC haplotypes, but occurred infrequently in semiallogeneic combinations. The experiments suggest that skin-induced allotolerance is maintained by an immunosuppressive mechanism, that is CyP-sensitive but resistant to rIL-2 treatment.

Different pretreatment schedules were applied to rat heart donors and their effect on heart interstitial dendritic cell content was observed using monoclonal antibodies and immunofluorescence techniques. Interstitial dendritic cell (IDC) numbers were correlated with the histology and survival of hearts transplanted into untreated allogeneic recipients. Hearts from AS donors pretreated with cyclophosphamide and total-body irradiation showed prolonged survival in DA recipients only when more than 95% of the graft interstitial dendritic cells were depleted. Reconstitution studies established that prolonged graft survival following donor pretreatment depended on the removal of bone-marrow-derived cells and that these were IDCs. These results suggest that the IDC is the most significant "passenger leucocyte", and that very small numbers of residual IDCs were still sufficient to cause rapid rejection. DA rat heart contained three times as many IDCs as the AS strain and DA hearts were much more difficult to deplete of IDCs. Pretreated DA hearts were rejected by AS recipients, although grafts with a lower IDC content resulted in an attenuated histological rejection response and a markedly decreased recipient lymphocytotoxin response. To be effective, donor pretreatment schedules had to be initiated 5 days prior to transplantation. Pretreatment 6 hr prior to transplantation failed to deplete IDC or prolong graft survival even in he weak (ASxDA)F1 to DA model. Pretreatment protocols used clinically have probably not removed IDCs adequately and the development of methods that deplete IDCs effectively could improve graft survival at no risk to the recipient.

A simple combine treatment with immunosuppressive agents permitting the growth of human tumor xenografts in conventional mice is presented. It consists of two simultaneous applications of 90 mg of procarbazine (PCH) per kg and 30 mg of cyclophosphamide (CY) per kg, alternating with two doses of 0.15 ml/10 g of antilymphocyte serum (ALS) for 4 days before grafting. No postgraft treatment is used. With a take rate of 100%, at 60 days after subcutaneous transplantation into male C3H mice, fragments of a human colon adenocarcinoma had grown (on the average) into cherry-sized tumors. Two osteosarcomas, an Ewing sarcoma, and a bronchogenic carcinoma were also studied and grew similarly. Tumors could be established in the three tested mouse strains but grew better in male animals. Synergy of PCH and CY and ALS depends upon the alternating sequence of both drugs and ALS. The histology of the colon carcinoma and the Ewing sarcoma was unchanged as compared to the tumors growing in nude mice. In contrast, the osteosarcoma developed in a more differentiated fashion in the immunosuppressed mice. The presented model may serve as a screening system for anticancer drugs.

The behavior of BALB/c myeloma transplants in (C57BL/10 X BALB/c)F1 hybrids was investigated. The F1 hybrids were less susceptible to the grafts than were the parental BALB/c hosts. Host susceptibility was X-ray and cyclophosphamide sensitive, but an immune hypothesis was not favored since no memory was demonstrable. Males were less susceptible than females, and rabbit antithymus serum had no effect on susceptibility.

A vascularized heterograft model using outbred strains of animals was developed by transplanting mouse hearts heterotopically into rats. With this species desparity rapid but not immediate graft rejection was observed, with a predictably narrow range of graft survival times. Morphological and immunohistological studies showed early deposition of fibrinogen and vascular and myocardial inflammation without prominent or consistent localization of either IgG or C3. Later more extensive changes were observed, and deposition of IgG and C3 were more prominent in the grafts. Pretreatment of the recipient with cyclophosphamide alone or cyclophosphamide plus antigen prolonged graft survival; however, no statistically significant difference was noted between these groups. Morphological and immunohistological alterations preceded clinical rejection, and tissue injury appeared to be mediated by humoral and cellular immune mechanisms and by the coagulation system. This model is potentially useful for the study of heterotransplantation.

4-hydroxycyclophosphamide (4HCy) inhibits T cell-dependent and direct B cell humoral responses of human lymphocytes in vitro, but causes a potentiation of the former in the lowest drug concentrations. Lymph node humoral responses are strongly sensitive, but those of spleen are relatively resistant to the drug. Although the induction of suppressor T cells is Cy-resistant, the expression of their function is sensitive. The suppressor cell function of antibody production appears to be more sensitive than the helper function.