A partial conditioning approach to achieve mixed chimerism in the rat: depletion of host natural killer cells significantly reduces the amount of total body irradiation required for engraftment.

BACKGROUND: Mixed allogeneic bone marrow chimerism induces tolerance to solid organ grafts. Although we previously reported that partially ablative conditioning with 700 cGy of total body irradiation (TBI) is sufficient to allow for bone marrow engraftment in mice, we determined that a minimum of 1000 cGy was required in the rat. Because T cells and NK cells are critical in bone marrow graft rejection, our purpose was to examine whether targeting of radioresistant NK cells and/or T cells in the recipient hematopoietic microenvironment would reduce the TBI dose required for engraftment of allogeneic rat bone marrow. METHODS: Wistar Furth rats received either anti-NK3.2.3 monoclonal antibodies on days -3 and -2, anti-lymphocyte serum on day -5, a combination of both or no pretreatment. TBI was performed on day 0 and rats were reconstituted with 100x10(6) T cell-depleted bone marrow cells from ACI donors. RESULTS: Engraftment of T cell-depleted rat bone marrow was readily achieved in animals conditioned with 1000 cGy TBI alone (12/12) and the level of donor chimerism averaged 89%. At 900 cGy TBI alone only one of eight recipients engrafted. In striking contrast, 11 of 12 animals pretreated with anti-NK monoclonal antibodies and irradiated with 900 cGy showed donor chimerism at a mean level of 41%. No further enhancement of bone marrow engraftment could be achieved when recipients were pretreated with antilymphocyte serum alone or antilymphocyte serum plus anti-NK monoclonal antibodies. Mixed allogeneic chimeras exhibited stable multilineage chimerism and donor-specific tolerance to subsequent cardiac allografts. CONCLUSION: Specific targeting of radioresistant host NK cells allows for a significant reduction of the TBI dose required for allogeneic bone marrow engraftment.

Alpha beta TCR+ T cells play a nonredundant role in the rejection of heart allografts in mice.

BACKGROUND: Although the transplantation of solid organs and cellular grafts is a clinical routine, the morbidity and mortality associated with immunosuppression is significant. This could be avoided by the induction of donor-specific tolerance. To develop targeted antirejection strategies and regimens to induce donor-specific tolerance, cell populations in the recipient-mediating rejection of solid organ and cellular grafts must be defined. In this study we examined the role of alpha beta-TCR+ cells in the rejection of allogeneic heart grafts, by use of knockout (KO) mice deficient in the production of alpha beta-TCR+ T cells. METHODS: C57BL/6-TcrbtmlMom (alpha beta-KO) and C57BL6/J (B6) recipient mice were transplanted with B10.BR/SgSnJ (B10.BR) or BALB/c heart allografts. Animals also received bone marrow from normal B10.BR donors, followed by donor-specific or third-party heart transplants. RESULTS: Naive B6 control mice rejected B10.BR and BALB/c grafts within 16 days. In striking contrast, B10.BR and BALB/c heart allografts were indefinitely accepted in unmanipulated alpha beta-KO mice. The immune responsiveness was restored after bone marrow transplantation from normal donors. After bone marrow transplantation major histocompatibility-disparate BALB/c third-party heart grafts were rejected, whereas donor-specific grafts were still accepted. CONCLUSIONS: alpha beta-TCR+ T cells play a nonredundant role in the rejection of heart allografts in mice. Bone marrow chimerism is associated with donor-specific transplantation tolerance.

Clinical applications of mixed chimerism.

Bone marrow transplantation (BMT) is currently a procedure that is associated with high morbidity and mortality. Thus, the clinical application of this technique is limited to the treatment of life-threatening hematopoietic malignancies. The morbidity and mortality of BMT is mainly related to graft-versus-host disease (GVHD), failure of engraftment, and toxicity related to fully myeloablative conditioning. GVHD can be prevented by T-cell depletion. However, T-cell depletion increases the risk of failure of engraftment. With the identification of a facilitating cell population that enables engraftment of hematopoietic stem cells across major histocompatibility barriers, the dichotomy between GVHD and failure of engraftment has been resolved. If one could overcome the toxicity of conditioning with the development of partially ablative conditioning strategies, BMT could be used for the treatment of a variety of nonmalignant diseases, as well as in the induction of donor-specific transplantation tolerance. This review outlines the development and advantages of partially ablative conditioning strategies and illustrates possible applications of the technique. Forty years ago E.D. Thomas discussed the potential of BMT for treating immunodeficiencies and for the induction of transplantation tolerance. BMT can be viewed as a natural form of gene therapy to replace a defective cell or enzyme with a functional and normally regulated one.

T-cell depletion of allogeneic bone marrow using anti-alphabetaTCR monoclonal antibody: prevention of graft-versus-host disease without affecting engraftment potential in rats.

Bone marrow chimerism may solve two major limitations in the transplantation of solid organs and cellular grafts: (1) the requirement for life-long immunosuppressive therapy, and (2) acute and chronic rejection. When untreated bone marrow is transplanted into major histocompatibility complex (MHC)-disparate rats, lethal graft-vs-host disease (GVHD) occurs in the majority of recipients. T-cell depletion using anti-CD3 and anti-CD5 monoclonal antibody (mAb) to avoid GVHD led to an increased occurrence of failure of engraftment. We previously identified a cellular population in mouse bone marrow that facilitates engraftment of highly purified hematopoietic stem cells (HSC) across complete MHC barriers. In light of the fact that facilitating cells have a CD8+/CD3+/TCR- phenotype and mostly coexpress CD5, we evaluated in this study whether T-cell depletion of rat bone marrow using anti-alphabetaTCR mAb would retain engraftment potential yet avoid GVHD. T-cell depletion of bone marrow was performed using anti-alphabetaTCR mAb and immunomagnetic beads. Recipients were conditioned with 1100 or 1000 cGy of total body irradiation and reconstituted with 100 x 10(6) T-cell depleted (TCD) MHC- and minor antigen-disparate bone marrow cells. Animals were monitored clinically and histologically for GVHD. Chimerism was assessed by flow cytometry. Immunomagnetic bead depletion resulted in a reduction of T cells from 1.92%+/-0.21% to 0.10%+/-0.04% of total bone marrow. T-cell depletion did not remove facilitating cells (CD8+/alphabetaTCR-/gammadeltaTCR-/NK3.2.3-) from bone marrow. Further, the engraftment potential of TCD bone marrow was not affected, as 100% of animals engrafted and high levels of donor chimerism were detectable. Animals reconstituted with TCD bone marrow showed no clinical evidence of GVHD and histology revealed none to minimal changes, whereas recipients transplanted with untreated bone marrow succumbed to severe lethal GVHD. T-cell depletion using antialphabetaTCR mAb and immunomagnetic beads selectively removes T cells from the bone marrow graft while sparing facilitating cells that are required for engraftment of allogeneic bone marrow across MHC barriers. Moreover, the cells required for engraftment of HSC do not produce GVHD.

Mixed allogeneic chimerism to induce tolerance to solid organ and cellular grafts.

Transplantation of solid organs and cellular grafts has become clinical routine in the last 30 years. However, the requirement for life-long immunosuppression is associated with infections, malignancies and end-organ toxicity. Moreover, the treatment fails to prevent chronic rejection. The induction of donor-specific transplantation tolerance would solve these problems, but has remained an elusive goal. One approach to achieve transplantation tolerance is through hematopoietic chimerism. This review outlines different concepts of hematopoietic chimerism focusing on macrochimerism. Mixed allogeneic chimerism, also known as macrochimerism, is defined as engraftment of hematopoietic stem cells achieved by bone marrow transplantation (BMT). It discusses the advantages and limitations of the BMT as well as approaches to overcome these limitations in the future.

A nonlethal conditioning approach to achieve engraftment of xenogeneic rat bone marrow in mice and to induce donor-specific tolerance.

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.

Effect of FLT3 ligand and granulocyte colony-stimulating factor on expansion and mobilization of facilitating cells and hematopoietic stem cells in mice: kinetics and repopulating potential.

We have previously identified a cellular population in murine bone marrow that facilitates engraftment of highly purified hematopoietic stem cells (HSC) across major histocompatibility complex (MHC) barriers without causing graft-versus-host disease. Here we investigated the effect of flt3 ligand (FL) and granulocyte colony-stimulating factor (G-CSF) on the mobilization of facilitating cells (FC) and HSC into peripheral blood (PB). Mice were injected with FL alone (day 1 to 10), G-CSF alone (day 4 to 10), or both in combination. The number of FC (CD8(+)/alpha betaTCR-/gamma deltaTCR-) and HSC (lineage-/Sca-1(+)/c-kit+) was assessed daily by flow cytometry. Lethally irradiated allogeneic mice were reconstituted with PB mononuclear cells (PBMC). FL and G-CSF showed a highly significant synergy on the mobilization of FC and HSC. The peak efficiency for mobilization of FC (21-fold increase) and HSC (200-fold increase) was reached on day 10. Our data further suggest that the proliferation of FC and HSC induced by FL in addition to the mobilizing effect mediated by G-CSF might be responsible for the observed synergy of both growth factors. Finally, the engraftment potential of PBMC mobilized with FL and G-CSF or FL alone was superior to PBMC obtained from animals treated with G-CSF alone. Experiments comparing the engraftment potential of day 7 and day 10 mobilized PBMC indicate that day 10, during which both FC and HSC reached their maximum, might be the ideal time point for the collection of both populations.

Baboon bone marrow transplantation in humans: application of cross-species disease resistance.

Xenotransplantation is a newly evolving field. A renewed interest has emerged coincidentally with the shortage of donor organs for transplantation. Bone marrow (BM) chimerism has been suggested as a potential strategy to induce tolerance to xenografts and control the immune response across a species barrier. Bone marrow transplantation (BMTx) displays unique features compared to solid-organ transplantation or transplantation of other cellular grafts. To achieve engraftment of the pluripotent hematopoietic stem cell, which generates all lineages of the hematolymphopoietic system, conditioning of the recipient (usually a combination of irradiation and cytoablative chemotherapy) is required. Once engraftment is achieved, graft function is stable and rejection does not occur, even without immunosuppression. On the other hand, the graft itself is able to generate an immune response against the host, resulting in graft-versus-host disease (GVHD). A newly recognized advantage to xenotransplantation is species-specific disease resistance. In terms of BMTx, important questions arise: Can xenogeneic BM generate a competent immune response across species barriers? Will cross-species GVHD occur? What are the possible applications to humans? This review addresses these questions. Problems emerging from xenogeneic BMTx are summarized and strategies for their solution discussed.

In vivo depletion of host CD4+ and CD8+ cells permits engraftment of bone marrow stem cells and tolerance induction with minimal conditioning.

BACKGROUND: Solid organ transplantation has become the preferred approach for the treatment of end-stage organ failure. However, the toxicity associated with the nonspecific immunosuppression essential to graft survival is substantial. Bone marrow transplantation (BMT) can overcome these limitations by the induction of donor-specific tolerance. The morbidity and mortality associated with fully ablative conditioning used to achieve engraftment has prevented the clinical application of BMT for induction of tolerance for solid organ transplantation. Although it was previously believed that fully ablative conditioning was essential to achieve engraftment, it has recently become apparent that partial conditioning may be sufficient to achieve chimerism and tolerance. The focus of this study was to characterize which cells in the host microenvironment must be eliminated for engraftment of MHC-disparate bone marrow to be achieved. METHODS: C57BL/10SnJ mice were depleted of CD4+, CD8+, or both cell types with monoclonal antibodies before irradiation with 300 centigray (cGy) and transplantation of 15 x 10(6) allogeneic (B10.BR) bone marrow cells. Two days after transplantation the animals were treated with 200 mg/kg cyclophosphamide. Animals were typed for chimerism at 28 days and monthly thereafter. RESULTS: The combination of CD4+ and CD8+ depletion resulted in multilineage engraftment in 76.5% of the animals at a level of 57.1 +/- 17.7%. The depletion of CD4+ cells alone was not sufficient to allow engraftment, whereas depletion of CD8+ cells alone was. CONCLUSIONS: T cells in the recipient's marrow space play an important role in hindering allogeneic engraftment in the mouse. The T-cell subset responsible appears to be CD8+ cells rather than CD4+ cells.

Bone marrow transplantation for therapy in autoimmune disease.

A variety of clinical and experimental reports have shown the interdependence between bone marrow and autoimmune diseases. Autoimmune diseases can be transferred as well as cured by bone marrow transplantation (BMT). The widespread application of this therapeutic approach is limited today by the morbidity and mortality associated with BMT, including failure of engraftment, graft-versus-host disease (GVHD) and the toxicity from lethal conditioning approaches. Mixed chimerism (with the advantage of superior immunocompetence of the host and a relative protection against GVHD) can be achieved with incomplete ablation conditioning regimens. BMT may provide a potential strategy to treat those autoimmune diseases for which today only symptomatic treatment is available.

Tolerance induction for islet transplantation.

Type I diabetes is a systemic autoimmune disease. Although transplantation of pancreatic tissues restores glucose homeostasis, grafts are affected by acute and chronic rejection as well as re-occurrence of autoimmune destruction. One newly recognized promising strategy to interrupt these detrimental processes is hematopoietic chimerism induced by bone marrow transplantation (BMT). The application of hematopoietic chimerism has three domains in the treatment of Type I diabetes mellitus: (1) tolerance induction to pancreas or pancreatic islet grafts; (2) prevention of the re-occurrence of autoimmune processes in the graft; (3) prevention of the onset of overt diabetes once the pre-diabetic state is clearly identified. Unfortunately, conventional BMT is associated with significant morbidity and mortality due to graft-versus-host disease (GVHD), failure of engraftment and lethal conditioning. The risk of these complications cannot be justified in the treatment of non-malignant diseases including Type I diabetes. This chapter will outline potential strategies to achieve hematopoietic chimerism without the risk of deadly complications. With these strategies, it may be possible to apply hematopoietic chimerism in the treatment of Type I diabetes, both to induce tolerance to islet allografts as well as to intervene and interrupt the autoimmune process in its early stages.

Correlation between graft-versus-host induced immunosuppression and host natural killer cell activity in small bowel transplantation.

The occurrence of graft-versus-host disease (GvHD) following small bowel transplantation (SBTx) can be tuned by the recipient's initial natural killer (NK) cell activity, which modifies the immunogeneic balance between donor and host immunocompetent cells. This study was aimed to investigate the role of host NK cells on the incidence and severity of GvHD following SBTx. Intraperitoneal administration of 50 microl ascites fluid of the highly specific anti-NKR-P1 monoclonal antibody (mAb) 3.2.3 into F1 recipient animals on three consecutive days prior to SBTx was performed to suppress NK activity in F1 hybrids. In vivo treatment with 3.2.3 mAb effectively depleted recipient NK activity for at least 10 days in spleens and mesenteric lymph nodes of F1 hosts. In contrast to nontreated F1 recipients, all 3.2.3 mAb-pretreated F1 animals suffered from severe signs of GvHD, and the mean survival time was decreased significantly from 16.0 +/- 0.9 days to 11.0 +/- 0.8 days (p < 0.01) in nontreated and NKR-P1-depleted F1 animals, respectively. Other sequelae included earlier onset of GvH manifestations, pronounced damage of primary and secondary lymphatic organs, substantial increase in spleen index, and lower CD4(+)/CD8(+ )ratios over the course of progressing GvHD. Our results underline the important immunoregulatory role of NK cells as a first defensive line acting on the alloreactivity of donor-derived immunocompetent cells in this model of solid organ transplantation.

Circumvention of natural killer cell and T-cell mediated allogeneic target killing with tacrolimus (FK506) in small bowel transplantation related graft-vs-host disease.

The role of simultaneous donor-specific transfusion of unprocessed cellular bone marrow (BM) together with solid organ transplantation, a postulated concept to achieve long-term graft acceptance, was investigated in an experimental setting of semiallogeneic transplantation of parental small bowel (SBTx) to F1 hybrids. The established graft-vs-host (GvH) model revealed that simultaneous transfer of SB/BM substantially enhanced GvH-mediated immune responses in recipient target organs, e.g. skin, gut, and liver. In comparison to isolated SBTx, animal survival decreased from 16.1 (+/- 0.9) to 10.1 (+/- 0.8) days after additional BM transfusion, P < 0.001. Severe tissue injury of GvH-susceptible target organs in the setting of simultaneous SB and BMTx was associated with significant changes in recruitment and tissue distribution of NKR-P1+ cells during the GvH-related proliferative immune response. Tacrolimus effectively suppressed these initial events and prevented recipient animals from clinically and histologically observed damage caused by GvH disease.

In vivo depletion of NKR-P1 positive cells in the recipient prior to small bowel transplantation enhances graft-versus-host disease (GvHD) in the rat.

Recent evidence for major histocompatibility complex (MHC) class I antigen-directed recognition mechanisms of natural killer cells (NKs) have revived interests in investigating non-adaptive immune responses in the framework of solid organ transplantation. A semi-allogeneic rat model of heterotopic small bowel transplantation (HSBTx) from male DA parental to male F1 hybrid rats (DA x LEW) was established to investigate the role of host NKs to attenuate graft-versus-host (GvH)-mediated immunosuppression and tissue injury. By use of anti-NKR-P1 monoclonal antibody (mAb) 3.2.3, host NKs were depleted effectively in vivo after triple intraperitoneal injection prior to HSBTx. In contrast to non-depleted animals, an initial lack of NK activity in F1 hosts significantly decreased the mean survival (P < 0.01) and substantially enhanced graft-versus-host disease (GvHD)-related damage to lymphoid and non-lymphoid target organs. These findings emphasize the important immunoregulatory role of host NKs during the early onset of GvHD.

Effect of a platelet-activating factor antagonist on pancreas perfusion after 24 h of ischemia.

Platelet-activating factor (PAF) is a strong mediator of inflammation that is present in many mammalian tissues and cell types. In the pancreas, PAF can be synthesized in acinar cells after stimulation with secretagogues. The present study uses a perfused porcine pancreas model to investigate the role of PAF in pancreatic ischemia and the effect of the PAF antagonist bepafant on pancreas preservation. Pancreata were preserved with or without bepafant, stored for 24 h at 4 degrees C, and then reperfused at 37 degrees C in a perfusion chamber. Reperfusions were significantly improved by the addition of bepafant. This was indicated by a significantly increased arteriovenous volume flow (16.54 +/- 1.88 ml/min versus controls 8.54 +/- 1.31 ml/min; p = 0.0068; bepafant, n = 7; controls, n = 12) and a reduced vascular resistance (p = 0.0068; bepafant, 1.95 +/- 0.22 mm Hg * min/ml versus controls 4.08 +/- 0.56 mm Hg * min/ml). Radioimmunological quantification of PAF in pancreatic tissue revealed that PAF levels remain unchanged during storage in a cold protective solution at 4 degrees C but increase significantly during surgical pancreas preparation under general anesthesia (from 142.1 +/- 21.2 to 368.8 +/- 52.5 pg/g; n = 15; p = 0.0007). The present study shows that bepafant improves pancreas preservation after cold ischemia. The beneficial effect might be explained by antagonizing inflammatory and vasoconstrictory responses to PAF synthesized during surgical pancreas preparation.